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Dr. Faria is associated with Department of Biochemistry (U38-FCT), Faculty of Medicine, University of Porto , 4200-319 Porto, Portugal.
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Dear Dr. Brown,
Below is the email address of Dr. Faria.
Tel./Fax: +351 22 551 36 24
You may refer to the article attached below.
Best.
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📢📢📢 Call for Papers: Memorial Issue to Prof. Kazuo Umezawa: A Noteworthy Biochemistry Educator
📅📅 Submission Deadline: 31 December 2024
 💫💫 Research Field: NF-κB, Immunology, Biochemistry, Cancer Research, Apoptosis
💡💡 Contact: christyhe99@gmail.com
Welcome your comments~
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Please send me a private message. I need to publish
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Hello!
I was wondering if anyone had a detailed protocol for measuring β-hexosaminidase activity in isolated lysosome fractions.
I will have 0.5ml fractions of lysosome isolate (after magnetic isolation).
Here is what I know so far:
  • samples: lysosome aliquots with and without Triton X-100 (1% final triton concentration) + whole cell lysates + cell culture supernatant + controls without sample
  • reaction buffer: 100 mM Na Citrat, 0.2% BSA, 1mM 4-MU-β-N-acetylglucosaminide
  • incubation: 10 min 37°C
  • stop solution: glycine/NaOH pH 10.4
  • fluorimeter: excitation 360nm, emission: 450nm
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Helena Milionis Do 25 uL of lysosomal eluate + 50 uL of substrate, incubate 30 min at least, then stop with 200 uL stop solution. This usually works for magnetically-purified lysosomes, but you can prolong the incubation time for sure. In some cases (exocytosis assays) I've increased it up to 24 hours.
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Serán empleados como antecedentes de una investigación con este propósito
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  • Complex Terminology: Biochemistry is filled with specialized terminology that can be overwhelming. Students may struggle to grasp the meaning of terms, which can hinder their understanding of the material.
  • Abstract Concepts: Many biochemical processes, such as metabolic pathways and molecular interactions, can be abstract. Visualizing these processes and understanding how they relate to nutrition can be challenging.
  • Integration of Knowledge: Biochemistry requires an integration of knowledge from various fields, including biology, chemistry, and physiology. Students may find it difficult to connect concepts from these disciplines and apply them in a nutrition context.
  • Mathematical Skills: Some areas of biochemistry involve quantitative analysis, such as enzyme kinetics or thermodynamics. Students who may not be confident in their math skills can find these aspects particularly daunting.
  • Laboratory Skills: Practical lab work is often a component of biochemistry courses. Students may struggle with laboratory techniques or data analysis, which can impact their overall understanding of biochemical principles.
  • Memorization: Biochemistry involves a significant amount of memorization, from amino acid structures to metabolic pathways. This can be overwhelming, especially when trying to retain a large volume of information.
  • Application to Nutrition: Connecting biochemical concepts to real-world nutrition applications can be challenging. Students may find it difficult to see how biochemical processes directly relate to dietary practices or health outcomes.
  • Dynamic Nature of the Field: Biochemistry is a rapidly evolving field with new discoveries and technologies. Keeping up with current research and understanding its implications for nutrition can be difficult.
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I have to estimate the growth of my bacterial cultures spectrophotometrically and I read articles of measurement at an OD of 600nm. Also what to do if the values exceed 1. What is the proper method for the measurement of the same.
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Different labs use different wavelengths to measure, usually from 590-650. They all work fine so it doesn't really matter so long as you consistently use the same. Traditionally 600nm has been used but not by everyone, I think because it matches the old color filters of Klett meters that used to be used.
When you get to optical densities above 1, the amount of light being scattered vastly exceeds the amount passing through, so the accuracy of the measurement goes way down. As the other responders said, the solution is to dilute your culture and take your reading so that it is below 1.
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Cure of cancer?, respectfully to whom it may concern:
If I found a cure of cancer, I’m wondering what I should do with it? I’m just coming out of a 40 days fast of only drinking maple syrup and pure lemon juice plus a little cinnamon powder and nothing else since I had unimaginable pain in my colon end. I figured out that although we humans can last for weeks on this diet /fast, cancer has no food on that and just goes away and the pain is gone too. I received this prescription by tuning in into the HeilstrOm and I figured out that it also enhances the reception of the HeilstrOm greatly, which brings great happiness too, as I describe in my book
Best regards, hope this helps someone
Chris K. Frueh
Independent Researcher
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@Seraphina Anderson good point
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In the identified case of familial desminopathy (T341P DES mutation in heterozygous state), the son has bradycardia, but the father did not have bradycardia. How can this fact be explained?
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The difference in clinical presentation between the son & the father, despite both having the T341P DES mutation in a heterozygous state, is dependent on other genetic variations, which can influence the expression & impact of the desmin mutation. These genetic modifiers can either exacerbate or mitigate the effects of the primary mutation, leading to different clinical outcomes. Epigenetic changes, like DNA methylation & histone modifications can also affect gene expression without altering the DNA sequence & can be influenced by environmental factors as well. Differences in lifestyle, diet, physical activity, exposure to environmental stressors can also impact how the mutation manifests. For example, factors like physical activity levels along with overall cardiovascular health can influence the development of bradycardia. The age at which symptoms appear can vary. The son might be at an age where the mutation's effects are more pronounced, while the father might not have exhibited symptoms at the same age or might have developed compensatory mechanisms over time. These factors highlight the complexity of genetic conditions and the importance of considering both genetic and non-genetic influences when assessing clinical presentations as such.
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I am measuring the specific activity of an enzyme under two different conditions using an enzymatic kinetic assay.
In one of my conditions, I know that the expression of the enzyme is highly upregulated. Therefore, even though I use equal amounts of sample (protein concentrations measured by BCA), I expect higher levels of the enzyme of interest in this condition.
In case I am investigating the activity per molecule of enzyme.
So, would it be appropriate to normalize the specific activity by dividing it by the enzyme expression data from a Western blot?
*Note: I subtract the background activity (before substrate addition) and non-specific activity (after addition of a specific inhibitor) to calculate the specific enzyme activity.
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Yes, if your goal is to measure and compare the catalytic activity per molecule of the enzyme (turnover number or kcat) in different samples, then divide the catalytic activities by the molar quantity of the enzyme in each sample.
If your goal is to measure and compare the amounts of enzyme activity in different samples, then divide the catalytic activities by the total protein concentrations in each sample.
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Dear researchers,
i would like to make a research on "high quality organic fertilizer production from biogas extracts"
looking forward to your sugessions
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Hafiz Muhammad Rizwan High-quality organic fertilizer production from biogas extracts involves several steps:
1. Separation: Separate the liquid (digestate) from the solid fractions of the biogas extract.
2. Dewatering: Remove excess water from the digestate through mechanical or thermal processes.
3. Pasteurization: Heat the digestate to eliminate pathogens and weed seeds.
4. Composting: Mix the digestate with carbon-rich materials and compost to stabilize the nutrients.
5. Granulation: Convert the composted material into a granular or pelletized form for easy handling and application.
6. Quality control: Test the final product for nutrient content, microbial populations, and contaminants to ensure high quality.
7. Fortification: Enhance the fertilizer with additional nutrients or microorganisms to improve its performance.
8. Packaging and storage: Store the fertilizer in a dry, cool place to maintain its quality until use.
This process produces a nutrient-rich, pathogen-free organic fertilizer suitable for various crops and soil types.
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I was wondering if it is possible to form a permanent open "ssDNA bubble" similar to a transcription bubble (>13 nucleotides) within E. coli. These criteria are important:
1. Open ssDNA bubble within replicable (in E. coli) genetic element. So no C-Traps under force.
2. No proteins, nucleic acids, or other toxic chemicals supporting the bubble. Can help during nucleation, but bubble has to be accessible for protein interaction.
3. Stable in bioorthogonal conditions. Physiological pH, salt, 37 °C, etc.
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Well, creating a semi-permeable transcription bubble can be challenging in the context of the structural stability of DNA as it tends to reanneal to its double-stranded form. Next is the concern of replication, which involves the fact that semi-permanent unwinding can potentially hinder the replication machinery from proceeding with DNA replication. Lastly, to maintain the transcription bubble to its semi-permanent unwound state, RNA polymerase is required to be halted in its activity at the transcription site, which could, in turn, lead to instability and interference in the replication of the plasmid. Considering these aspects, I believe three probable yet theoretical strategies can be adopted in this regard. First is genetically engineering a modified RNA polymerase, which can maintain the plasmid DNA at its single-stranded state by getting associated at a precise plasmid location without hindering the transcription process. Second is implementing genetically engineered single-strand binding (SSB) proteins, which can keep the plasmid DNA at its unwound state without interfering with RNA synthesis. Lastly, chemical molecules such as intercalating agents are introduced, which can develop proximal unwinding by being inserted at the nitrogenous base pairs of plasmid DNA; Molecules that are enhancers or activators of helicases; Hydrogen bond destabilizers like Di-Methyl Sulfoxide (DMSO), Urea or Formamide which can perform denaturation of double-stranded DNA; Cross-linking agents like Psoralens which forms covalent cross-linkages between single-stranded DNA molecules and DNA or RNA polymerases; Ligands which associate with single-stranded DNA such as Peptide Nucleic Acids (PNAs) and nucleic analogs which stabilizes the single-stranded structures of DNA; Alkylating agents such as Nitrogen and Sulfur derivatives of Mustard gas, Ethyl Methanesulfonate (EMS), Methyl Methanesulfonate (MMS), N-Nitrosoureas and Temozolomide. Nevertheless, besides being hypothetical, all these strategies have cons of their own.
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I was performing estimation of Total Soluble Sugar content in bacterial cultures by Anthrone method. Out of 9 samples, only one sample showed positive reading while others had a negative reading in the UV-vis spectrophotometer. What does this denote.
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Swapnil Srivastava then, you could have carbohydrates in your samples that do not form furfural in the dehydration step, so the response is negative for them.
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In My article: The Extended Pedersen hypothesis (published, February 1988 Clinical Physiology and Biochemistry 6(2):68-73,) there is mistake of the name of first Author: it is not Catherine Macfarlane, but Campbell M. Macfarlane.
Can you please correct it?
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Nicholas Tsakalakos You can do that yourself by editing your publication and add Campbell M. Macfarlane as your right co-author; this usually works. Of couse, you have to delete Catherine Macfarlane, as your false co-author. Success !
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I have extracted essential oil from a medicinal plant and want to make several concentrations for some biological activities (Antidiabetic assay). How can i prepare the several concentrations to determine the IC50.
What will be the solvent for the dilution of the essential oils?
Your kind help in this regard will be highly acknowledged.
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To assess the biological activity of essential oils, such as their antidiabetic properties, it is necessary to prepare several concentrations to determine the half-maximal inhibitory concentration (IC50). The following steps outline the process for diluting essential oils and the choice of solvent:
Dilution of Essential Oils for Biological Assays
1. Selection of Solvent:
The choice of solvent is critical for the solubility of essential oils and the accuracy of the biological assay. For essential oils, organic solvents such as dimethyl sulfoxide (DMSO), ethanol, or methanol are commonly used. These solvents help to dissolve the hydrophobic components of essential oils efficiently. Water can also be used, but it often requires an emulsifier or surfactant to achieve proper dissolution.
2. Preparation of Stock Solution:
- Begin by preparing a concentrated stock solution of the essential oil. Typically, a stock solution of 10 mg/mL or higher is prepared in the chosen solvent. For instance, dissolve 100 mg of essential oil in 10 mL of DMSO.
- Ensure thorough mixing to achieve a homogeneous solution. This can be done using a vortex mixer or by gentle heating if necessary (within the stability limits of the essential oil).
3. Serial Dilution:
- To create a range of concentrations, perform serial dilutions of the stock solution. For example, if the desired concentration range is from 0.1 mg/mL to 10 mg/mL, you can follow these steps:
1. Take 1 mL of the stock solution (10 mg/mL) and add it to 9 mL of solvent to obtain a 1 mg/mL solution.
2. Take 1 mL of the 1 mg/mL solution and add it to 9 mL of solvent to obtain a 0.1 mg/mL solution.
3. Repeat this process as necessary to achieve lower concentrations.
4. Assay Preparation:
- Once the different concentrations are prepared, they can be used in biological assays. For antidiabetic assays, in vitro methods such as enzyme inhibition assays (e.g., alpha-glucosidase or alpha-amylase inhibition) are common.
- In these assays, add varying concentrations of the essential oil solution to the reaction mixture and measure the inhibitory effect compared to a control without the essential oil.
Determination of IC50
- The IC50 value represents the concentration of the essential oil required to inhibit 50% of the biological activity. To determine this, plot the percentage inhibition against the logarithm of the essential oil concentration.
- Fit the data to a suitable model, such as the logistic regression model, to calculate the IC50 value using software tools like GraphPad Prism or similar.
References
For accurate scientific referencing, you can refer to the following sources:
- Nazzaro, F., Fratianni, F., De Martino, L., Coppola, R., & De Feo, V. (2013). Effect of essential oils on pathogenic bacteria. Pharmaceuticals, 6(12), 1451-1474.
This paper discusses the methodologies for evaluating the biological activities of essential oils and provides insights into solvent choices and dilution techniques.
- Pandey, A., & Tripathi, S. (2014). Concept of standardization, extraction, and pre-phytochemical screening strategies for herbal drug. Journal of Pharmacognosy and Phytochemistry, 2(5).
This article provides details on extraction methods and solvent selection for essential oils.
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It is known that patients with desminopathy often die from pneumonia. Have pathomorphological studies of the lungs been performed in patients with desminopathy?
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Dear Sagar Nanaso Salunkhe, thank you very much for your detailed answers!
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Hi! I'm doing a renilla luciferase assay with coelenterazine and HEK293T cells transfected with AP-1 renilla luciferase. I'm wondering if the cells need to be lysed prior to adding coelenterazine and measuring the luminescence. Coelenterazine is cell-permeable, so I'm wondering if/why the lysis step would be necessary.
I understand that the proteins would be more exposed--would the luminescence be not as great through the cell membrane if we didn't do the lysis?
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Sindhu Kondath ... Dual Luciferase assay is not to test cell viability. among other things, it is mainly used to test DNA repair capability. For cell viability, I think MTT or WST-1 would be the best assay
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We are researching the conjugation of various antibodies to quantum dot microspheres and europium microspheres. The sustainability results have been unsatisfactory. Which stabilizing buffers do you recommend for stabilizing the conjugation step?
Best regards
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The appropriate stabilizing buffer for conjugating quantum dots (QDs) to antibodies is critical for ensuring the stability and functionality of both the QDs and the antibodies. The buffer utilized is determined by a number of factors, including the type of QDs, the conjugation chemistry, and the antibodies used. However, some general rules can help you choose an adequate buffer:
  1. pH: The buffer should maintain a pH that is compatible with both the quantum dots and the antibodies. Typically, a pH range of 7.0 to 8.5 is used for conjugation reactions.
  2. Buffer Components:Phosphate-buffered saline (PBS): Commonly used because it is biologically compatible and maintains pH stability. A typical concentration is 10 mM PBS with 150 mM NaCl. HEPES: Another buffer that is often used, especially for reactions that are sensitive to phosphate. It maintains a stable pH in the range of 7.2 to 7.5.
  3. Additives:Bovine Serum Albumin (BSA): Often added at concentrations of 0.1% to 1% to prevent nonspecific binding and to stabilize proteins. Tween 20 or Triton X-100: Low concentrations (e.g., 0.01% to 0.1%) of non-ionic detergents can help reduce nonspecific binding without significantly affecting the conjugation process. Glycerol: Can be added up to 10% to improve stability during storage.
  4. Chelators: Avoid using chelators like EDTA or EGTA if the conjugation involves metal-affinity interactions, as they can chelate metal ions essential for the quantum dot stability.
  5. Reducing Agents: Avoid using reducing agents such as DTT or β-mercaptoethanol during conjugation if the chemistry relies on disulfide bonds, as they can disrupt the bonds necessary for the conjugation.
Example Buffer Composition
A commonly used stabilizing buffer for conjugation might look like this:
  • 10 mM PBS, pH 7.4
  • 0.1% BSA
  • 0.05% Tween 20
Conjugation Chemistry Considerations
  • Carbodiimide Chemistry: Often used for conjugating carboxyl-functionalized QDs to amine groups on antibodies. Buffers like MES (2-(N-morpholino)ethanesulfonic acid) at pH 5.0 to 6.0 are commonly used for the activation step, followed by conjugation in PBS.
  • Maleimide Chemistry: Used for thiol groups on antibodies. Buffers should be free of primary amines and free thiols.
Final Tips
  • Buffer Exchange: Ensure that the antibodies and QDs are in the same buffer system before starting the conjugation to avoid precipitation or aggregation.
  • Optimization: Perform small-scale trials to optimize the buffer conditions for your specific QD and antibody combination.
  • By carefully selecting and tailoring the stabilizing buffer, you can successfully attach quantum dots to antibodies, assuring the stability and usefulness of the bioconjugates that result.
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I have purchased Acetylcholiesterase from Electrophus electricus (electric eel), C3389-2KU, following details are written on the enzyme vial.
Type-VI S lyophilized powder 200-1000 Units/mg protein, 374 units/mg solid, 610 Units/mg protein, 5.3 mg/solid.
i want to prepare 0.28 U/ml working solution, please help me with the preparation of stock solutions and the calculations.
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The answer depends on whether you plan to use the whole sample or weigh some out.
If you plan to use the whole sample of 5.3 mg, you can calculate the number of units: 5.3 mg x 374 U/mg = 1982 units. If you were to dissolve this at 0.28 U/ml, you would end up with 1982 U/(0.28 U/ml) = 7078 ml. So instead, you would make, for example, a 1000X concentrated stock solution of 7.078 ml, or even a 10,000X stock solution of 708 µl. This could be stored as small aliquots in the freezer and diluted as needed.
If you have access to an analytical balance capable of accurately weighing out 1 mg, you could use a portion of the sample. For example, starting with 1.0 mg, you would dissolve it in (1/5.3) x 7.078 ml = 1336 µl for a 1000X solution or 134 µl for a 10,000X solution.
You should dissolve it in a suitable buffer to retain its activity.
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What is the difference between absorption and adsorption?
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The main difference is that while absorption involves the mass transfer of particles into another material (one substance absorbing another), adsorption takes place with the adhesion of particles onto the surface of a substance. absorption is the process in which a fluid dissolves by a liquid or a solid. In the case of Adsorption, the atoms, ions, or molecules from a substance adhere to a surface of the adsorbent
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Hi, are there other methods beside Western Blotting to detect a specific protein? I‘ve isolated mitochondria and want to show that hPar17/Pin4 is present in mitochondria. The Western Blots didn’t work well, because of the low amount of sample or maybe low expression levels of hPar17 (the Abs work). I want to detect the protein without overexpressing it if possible. I isolate the mitochondria from human cardiac myocytes. Do you have any ideas on how to show the presence/expression of my protein in mitochondria? Thank you very much!
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Cell staining is possible, especially if you want to co-localize your protein. However, insufficient sensitivity will likely be an issue. MS would indicate presence of your protein, but that's about it. If I were you I would seriously consider protein tagging and/or overexprexsion to start off. You could then perform coIP, proximity based assays etc...Good luck!
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Background Biochemistry
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we can help with theoretical analysis (Computational) in your publication. can you plz put forward your research question?
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Hi, I am looking to perform an analysis of Br/ KBr in samples of water sources to see if the levels are safe. Due to budget and resource limitation at the stage, I would prefer a method that considers these factors.
Kindly help.
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KBr is 100% ionized and in aqueous solution the counter ion is anything that is a cation (positively charged) that is floating around. If your buffer contains K+ as the most common cation,then [Br-] = [KBr].
I have used the colorimetric assay previously given.
Bowen H.J.M. The determination of chlorine, bromide and iodide in biological material by activation analysis. Biochem. J. 1959; 73: 381
It is easy to use and cheap, despite using a gold salt because you don't use much.
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I am planning to purify the mononucleosome for cryoEM study. I have seen, people assemble the nucleosome from recombinantly expressed histones and then providing DNA sequence to it. Isn't this possible to islolate nucleosome and after enzymatic cleavage, followed by SEC chromatography for the cryoEM study ? I understand that there could be a population of others but it could be more relevant. Please suggest.
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SEC is an excellent technique for discriminating between monomer, oligomer, and aggregated forms of a target protein. Resin characteristics.
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Hello,
My lab recently acquired a combination ORP electrode, with the refrence being Ag/AgCl and the working electrode being platinum.
We are hoping to use this electrode to measure the reduction potential of buffers prepared using biologically active compounds, such as GSH and GSSG and probe redox active systems by artificially setting the potential.
Recently, I was trying to validate the electrode by preparing 1 mM total concentration solutions of varying ratios of GSH and GSSG. This was done as, to my understanding regardless of concentration the ratio of Ox vs Red determines the potential value of the solution via the Nernst Equation. However the readings I got were all positive, and nowhere close to the expected potential, even when correcting for the electrode difference between Ag/AgCl and SHE.
Secondly, in 1x PBS pH 7.4, I added increasing amounts of BME up to 1 M and got an exponential decay like curve asymptotically approaching ~-120 (SHE) mV.
I am having trouble making sense of these results, namely the GSH vs GSSG ratio, and why the readings would not follow the nersnt equation.
Can anyone explain how to use these ORP electrodes, and where I may be going wrong in these experiments? All the information I can find online are referring to waste water treatment.
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Dear Darius Chernitsky,
I see two serious mistakes in your reasoning concerning the Nernst equation as applied to the measurement of the redox potential, specifically, in the glutathione (GSH/GSSG) system.
First. The statement that “regardless of concentration the ratio of Ox vs Red determines the potential value…” is wrong, since the Nernst equation is defined in terms of activity, not concentration. (In simple terms, intermolecular interactions reduce the effective concentration of the particles in solution so that from the "viewpoint of an electrode", the perceived concentration is smaller than the actual concentration.) This means that activity coefficients both of the components of the redox couple should be taken into account. The data on activity coefficients in solutions of GSH/GSSG may not be available, especially in buffer systems in which you are going to measure the potentials.
Second. The Nernst equation is applicable for equilibrium conditions, both “inside” the redox system and between the electrode and the redox components in solution. From the fact that the equilibrium between GSH and GSSG in biochemical systems is established rapidly (due to the involvement of enzymes) does not follow that this will be so in model solutions. It is natural to expect the appearance of kinetic limitations. Thus, the simple solution to the problem that you hoped for is hardly possible.
Regards,
Rouvim Kadis
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I am attempting to synthesize a schiff base ligand by reacting 5-hydroxytryptamine (HCl salt) and pyridoxal (HCl salt) in methanol, but the final product is not crystallizing in the methanol to be recrystallized. The product is originally a sticky brown oil that solidifies as a whole. What should I try differently? Sulfuric acid catalyst and maybe glacial acetic acid? Anything else?
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I would throw in 2 equivalents of an organic base such as TEA (triethylamine) to trap the HCl. This would result in deprotection of the primary amino group (protected by HCl) of hydroxytryptamine and free pyridoxal from its aminium salt. Triethylammonium chloride would remain in solution while the Schiff product may precipitate out. Filter off, wash twice with methanol and dried in oven.
In case of inconclusive purity by TLC, redissolve precipitate in DCM, wash twice with water, dry over MgSO4 and column if necessary.
An alternative route would be to deprotect both reagents separately before the Schiff reaction. To this end, dissolve a quantity of reagent in water, adjust pH to 8-9, the precipitate that form is filter off and dried.
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I tried glycine buffer but the yeast cells acidify the buffer so the pH goes down to 7-6 overnight. I need something that will stay around 9 and that isn't toxic to the cells.
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To maintain a high pH (pH 9) for yeast cell suspensions, you can use a buffer that is effective in this pH range. Tris buffer is commonly used for maintaining alkaline pH and may be suitable for your needs. Here's how you can prepare Tris buffer at pH 9:
  1. Tris Base (Tris(hydroxymethyl)aminomethane): Dissolve Tris base in distilled water to make a 1 M stock solution. The molecular weight of Tris base is 121.14 g/mol, so to make a 1 M solution, dissolve 121.14 g of Tris base in 1 liter of water.
  2. Adjust pH: Adjust the pH of the Tris solution to 9 using concentrated hydrochloric acid (HCl) or concentrated sodium hydroxide (NaOH). Use a pH meter or pH strips to monitor and adjust the pH as necessary.
  3. Final Dilution: Once the desired pH is achieved, dilute the Tris solution to the desired concentration for your experiment. Common working concentrations for Tris buffer range from 10 mM to 100 mM.
  4. Sterilization: Filter-sterilize the Tris buffer using a 0.22 μm membrane filter to remove any particulate matter and microorganisms.
  5. Storage: Store the Tris buffer at room temperature (if using within a few weeks) or at 4°C for longer-term storage. Avoid repeated freeze-thaw cycles.
Tris buffer is widely used in biological research and is generally compatible with yeast cell suspensions.
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Can a published journal article be submitted to conferences?
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It is quite common to present talks or posters on the basis of previously published papers. However, care must be taken when contributing to the proceedings of the conference so as not to infringe the copyright of the journal's publisher.
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Most of the researchers use to teach at university. In some careers, professionals who exert their profession without doing research share teaching spaces. When I was a chemistry student, 100% of my teachers were researchers ranging from PhD candidates to experts in their respective fields. While it may seem logical for researchers to be the best candidates to teach in fields such as chemistry or biology, what about healthcare-related fields like medicine, pharmacy, or biochemistry? Who is better suited to lead a class, a researcher or a professional, or both, each one in different subjects? We can distinguish between basic and clinical subjects. I am interested in hearing your thoughts on this matter.
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Being a proficient researcher doesn't necessarily equate to being a better professor. While research expertise can enhance teaching by bringing current knowledge and real-world applications into the classroom, effective teaching requires distinct skills such as communication, empathy, and the ability to engage students.
A good professor balances both research and teaching responsibilities, tailoring their approach to meet the needs of their students while contributing to their field through research. However, being a successful researcher doesn't guarantee effective teaching, as teaching requires its own set of abilities and dedication.
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In all academic sources, sucrose is identified as α−glucose (1-->2) β−fructose. However, I cannot find any explanation anywhere as to why the fructose monomer has to be in the β configuration. Maltose has both α and β anomers, same for lactose. Even trehalose, another non-reducing disaccharide with glycosidic linkage between two anomeric carbons, has α-α, α-β, and even β-β anomers. Why is sucrose special? And is there a disaccharide out there that has α−glucose (1-->2) α−fructose configuration?
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It's because in sucrose the anomeric C atoms of glucose and fructose are both involved in the glycosidic bond. That's why they can't change their configuration without breaking the glycosidic bond. In reducing disaccharides like maltose the anomeric C of the "second" glucose is "free" (hemiacetal only). Therefore, the open-chain form and all possible configurations (alpha, beta, furanose, pyranose) of the second sugar are available without breaking the glycosidic bond.
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A patient with desminopathy (mutation Thr341Pro DES in a heterozygous state) with the progression of the disease has a decrease in taste and smell, immunosuppression, and an increase in IgA in the blood.
Oddly enough, but all this is characteristic of infections, including viral ones. For example, it is known that if the hepatitis C virus is not treated, then death will occur in 20 years.
In the identified case of late onset desminopathy, muscle weakness manifests itself at the age of 30, and death occurs 20 years after the onset of the disease.
Could the desmin mutation in myofibrillar myopathy be caused by an infection?
Perhaps the infection contributes to the progression of desminopathy?
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Dear Esteemed Colleague,
Greetings. I trust this message finds you deeply engaged in your research and seeking answers to complex questions within the realm of genetics and molecular pathology. Your inquiry regarding the potential role of infection in causing desmin mutations in myofibrillar myopathy is both intriguing and indicative of a keen scientific mind exploring the multifaceted nature of genetic disorders.
To address your question with the precision and clarity it deserves, it is crucial to first understand the nature of myofibrillar myopathies and the role of desmin within this context. Myofibrillar myopathies are a group of neuromuscular disorders characterized by the progressive weakening of muscles and the disintegration of muscle fibers at a cellular level. Desmin, a type of intermediate filament protein, plays a pivotal role in maintaining the structural integrity and function of muscle cells. Mutations in the DES gene, which encodes the desmin protein, are directly linked to certain forms of myofibrillar myopathy.
The genesis of these mutations, particularly those affecting the desmin protein, is primarily genetic, resulting from inherited or de novo mutations in the DES gene. These mutations lead to the production of an abnormal desmin protein, which disrupts the normal architecture of muscle cells, leading to the symptoms associated with myofibrillar myopathy.
Addressing the specific question of whether an infection could cause desmin mutations, it is essential to differentiate between the origins of genetic mutations and factors that may exacerbate the phenotype of a genetic disorder. Genetic mutations, including those affecting the desmin gene, arise from alterations in the DNA sequence. These alterations can be inherited from parents, occur spontaneously during DNA replication, or be induced by certain environmental factors, such as exposure to specific chemicals or radiation. Infections, while capable of causing a wide array of health issues, do not directly induce genetic mutations in the DNA sequence of the genes like DES. However, it is conceivable that certain infections could exacerbate the clinical manifestations of myofibrillar myopathy in individuals already predisposed or carrying a desmin mutation, by stressing the muscular system or triggering inflammatory responses that may further compromise muscle function.
In conclusion, while infections can have significant impacts on overall health and may interact in complex ways with genetic disorders, the mutations in the DES gene that cause myofibrillar myopathy are not directly caused by infections. The mutations are genetic in origin, and the relationship between infections and the severity or progression of myofibrillar myopathy would be more accurately viewed through the lens of infection exacerbating pre-existing conditions rather than causing the genetic mutation itself.
I hope this elucidation addresses your inquiry comprehensively. Should you have further questions or require additional clarification, please feel free to reach out.
Warm regards.
This protocol list might provide further insights to address this issue.
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I need some suggestions for articles recently focused on the computational design of proteins, as well as the evaluation of the designed proteins by assessing various properties or values. If anyone is interested in this field and has read articles on this topic, please don't hesitate to share your suggestions below
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Hi, you can read some papers:
1. Computational Protein Design: Advances in Algorithms and Biophysical Validation
This article discusses recent advances in computational protein design algorithms and methodologies, focusing on biophysical validation techniques to assess the stability, structure, and function of designed proteins.
2. Machine Learning Approaches for Protein Design: Recent Advances and Future Directions
This review article explores the application of machine learning techniques in protein design, including deep learning approaches, and discusses methods for evaluating designed proteins using molecular dynamics simulations and experimental validation.
3. Rational Protein Design: Computational Approaches and Experimental Validation
This paper presents computational strategies for rational protein design, highlighting methods such as Rosetta and FoldX, and provides insights into experimental techniques for validating designed proteins through biophysical assays and structural characterization.
4. Multi-Objective Optimization in Computational Protein Design: Balancing Stability, Function, and Specificity
This article focuses on multi-objective optimization strategies in computational protein design, aiming to balance various design objectives such as stability, function, and specificity. It discusses evaluation metrics and experimental validation approaches for assessing designed proteins.
5. Integration of Structural Bioinformatics and Machine Learning for Protein Engineering
This study presents an integrated approach combining structural bioinformatics and machine learning for protein engineering, emphasizing the importance of accurate protein structure prediction and molecular dynamics simulations in evaluating designed proteins.
6. High-Throughput Screening Platforms for Computational Protein Design
This review article surveys high-throughput screening platforms used in computational protein design, including methods based on protein-protein interactions, enzyme assays, and cell-based assays, and discusses their applications in evaluating large libraries of designed proteins.
7. Protein Design in the Age of Synthetic Biology: From Theory to Experiment
This paper explores the intersection of protein design and synthetic biology, discussing computational tools for designing novel protein functions and experimental techniques for characterizing designed proteins in living systems.
These articles provide insights into recent advancements in computational protein design and offer strategies for evaluating the properties and functions of designed proteins through computational and experimental approaches.
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Dear Researcher Gate,
I inquired about adding a reviewer to the submitted manuscript in Biochemistry can you help me with this?
Thanks
maha saad
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Dear Maha Maki In addition to what is indicated by Michael J. Benedik you can come up wit suggestions by:
-Use names you know in your field of expertise
-Use names of those who published relevant papers in your field
-Use names of the publications you cited
Nowadays all the relevant contact info can be found in their publications.
Best regards.
PS. You do not have to know these people personally, the journal just want to increase their network of potential peer-reviewers with people you estimate to be able to review your manuscript.
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I am currently learning about PyMol to utilize in my project. I used PyMol to visualize potential H-bond interactions in specific amino acid residues. However, I have discovered that Arg465 and Ser461 show a distinct interaction, as shown.
Please help identify this interaction.
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The broken yellow line with the distance indicator (6.2) looks like a simple distance monitor which you generate with a "measure" command, although I do not know how you generated the blue tubes around it. At 6.2Å, the Ca-Ca distance indicated by the broken line is far larger than the sum of the carbon Van der Waals radii (3.4Å). It is just about short enough that you might classify the contact as a solvent excluding contact (hydrophobic interaction)
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An unopened Sigma-Aldrich (P4557) phenol solution bottle was shaken (prior to the addition of the Equilibration Buffer) and a gel-like layer formed at the bottom of the bottle. The upper phase is still liquid. The bottle was shaken briefly after the phenol solution was taken out of +4 C. What should be done? Should it be heated in order for it to return to liquid?
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All you need to do is to heat it up slightly, but not with real flame, do that by gently stirring it until it returns to liquid. If this does not work, contact the supplier/company for assistance. Thank you.
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Hi,
I am looking for build up a consensus or a group in clinical biochemistry for mutual exchange in scientific idea, research protocols, cooperation in proposal preparation, sharing in books, Research articles and reviews.
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I am in fir such collaboration. Cant wait to get started
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It is known that in the early stages of desminopathy the muscles most often affected are: Semitendinosus, Gracilis and Sartorius. What is the reason for the damage to these particular muscles?
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Desminopathy, also known as desmin-related myopathy (DRM), is a rare genetic muscle disorder that affects the protein desmin. Desmin is an essential component of the intermediate filaments that provide structural support within muscle cells. Mutations in the DES gene, which codes for desmin, lead to disruptions in the normal structure and function of muscle fibers.
The muscles you mentioned - Semitendinosus, Gracilis, and Sartorius - are often affected at the onset of desminopathy due to their specific characteristics and biomechanical roles.
1. Semitendinosus: The semitendinosus is one of the three hamstring muscles located in the back of the thigh. It plays a key role in knee flexion and hip extension. The semitendinosus muscle is frequently involved in desminopathy due to its high proportion of slow-twitch muscle fibers, which are more vulnerable to desmin-related abnormalities.
2. Gracilis: The gracilis muscle is a long, thin muscle located in the inner thigh region. It is involved in hip adduction and knee flexion. Similar to the semitendinosus, the gracilis muscle also consists of a high proportion of slow-twitch muscle fibers, making it susceptible to desmin-related abnormalities.
3. Sartorius: The sartorius muscle is a long, strap-like muscle that runs diagonally across the front of the thigh. It plays a role in hip and knee flexion and also assists in thigh rotation. The sartorius muscle is affected in desminopathy due to its similar composition of slow-twitch muscle fibers.
The predilection for these specific muscles in desminopathy may be attributed to their fiber type composition and the mechanical stress they experience during certain movements. However, it is important to note that desminopathy can affect other muscles as well, and the degree and pattern of muscle involvement may vary among individuals with the same genetic mutation.
It is advised to consult with a medical professional or genetics specialist for a more accurate assessment of muscle involvement and management of desminopathy.
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In biochemistry, most work is done at room temperature. Yet, basic thermodynamics tells us that affinities often change with temperature, in positive or negative directions depending on the entropy/enthalpy contributions. Thermal transitions can occur which both quantitatively and qualitatively change the behavior of the molecules of study. In enzymatic reactions rate-limited by diffusion-mediated product release, the increased rate of diffusion could increase the rate of product release above the rate of the chemical step, such that the chemical step becomes rate-limiting. If one discovers a compound that potently inhibits this enzyme at 25C, it may have reduced effect at 37C, or none at all. Likewise, if an enzyme is predominantly dimerized at 25C based mostly on enthalpic contributions, this dimer may not even exist at 37C. Screening compounds against the dimer may be of little relevance to the situation in vivo. The converse could happen if dimerization is entropically driven. Temperature-dependent changes in solution properties can also obscure the relevance of 25C results to 37C, such as viscosity.
I welcome everyone's two cents.
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I don't think it's true that "Biochemistry experiments are done at room temperature, not 37C." They are often done at 37C. However, I do most of my enzyme assays at room temperature because I usually work with 384-well microtiter plates, and it is difficult to establish a consistent elevated temperature across the whole plate on a short time scale. It is important to report the temperature in any publications. Surprisingly, this detail is often forgotten in Methods sections. Also, "room temperature" should be defined as a specific temperature or range of temperatures.
If a detailed mechanistic understanding of the biochemical mechanism is being investigated, it is very important to be clear about the temperature. Moreover, varying the temperature can provide useful information about the chemical mechanism, such as the activation energy of the reaction.
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Here is an excerpt from Kollmann et al. (2020, J. Physiol.):
"This ring was placed in a recording chamber continuously perfused with 37°C aerated Hepes solution containing (in mM) 136 NaCl, 10 glucose, 5 KCl, 10 Hepes, 1.2 MgCl2, 2.5 CaCl2 (pH 7.40) at a rate of 11 ml min-1. Hypoosmolality of the Hepes solution was established by reduction of the NaCl content to 33 mM (94 mOsm kg-1 H2O), 58 mM (144 mOsm kg-1 H2O) or 83 mM (194 mOsm kg-1 H2O)."
For validation, I tried to calculate the osmolality by myself. For example, when the NaCl content is changed to 33 mM, the osmolality should be
33*2 (NaCl)+10*1 (Glucose)+5*2 (KCl)+10*1 (Hepes)+1.2*3(MgCl2)+2.5*3(CaCl2)
which gives 107.1 mOsm/L, i.e., 107.1 mOsm/kg. But 107.1 is largely different from what the author stated, namely 94 mOsm/kg.
When the NaCl content is changed to 58 mM or 83 mM, the osmolality should increase by (58-33)*2=50 mM, or (83-33)*2=100 mM. This is consistent with the author's calculation, i.e., 144-94=50 mM, or 194-94=100 mM. So, my calculation is correct at least in terms of NaCl. But how can I calculate the contributions of the remaining solutes correctly?
Reference: Kollmann, P. et al. Submucosal enteric neurons of the cavine distal colon are sensitive to hypoosmolar stimuli. J. Physiol. 598, 5317–5332 (2020).
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Your calculations are correct if and only if you assume an osmotic coefficient of 1.0 for each solute. The manuscript doesn't describe how the osmotic coefficients were determined or if tabulated values were used, but does describe the use of an osmometer in at least one instance, so it is possible that these were experimentally determined, but this isn't clear from the text.
In any case, details about how the solutions were prepared are described in enough detail to repeat them accurately. I suppose you would need to find tabulated values for osmotic coefficients to arrive at the same number as the authors, or alternatively, you could use an osmometer to determine the NaCl needed to adjust your own solutions to the same osmotic pressures/osmolality.
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Is it correct to filter the extracts we obtain with Whatman filter paper and use them directly in experiments to investigate the bioactivity of phytochemical substances? Or is it correct to first centrifuge the extracts we obtain, then filter the supernatant and use them in experiments?
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You should first carry out a rough clarification of the extract by simply decanting, followed by a filtration step by using a six to seven layered muslin cloth. Then you may include a centrifugation step which may be required if the powder is too fine to be filtered.
I have attached a book below which may be helpful.
Best.
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Some (but not all) DNA polymerases such as Klenow fragment, Taq, and Phi 29 DNA polymerase can catalyze strand displacement synthesis. This is evidenced by opening of molecular beacons and Loop Mediated Isothermal amplification (LAMP).
in strand displacement synthesis, the primer strand is extended using the template strand and each nucleotide incorporated displaces the 3rd strand that was originally annealed to the template?
where is the third DNA strand? the one being displaced? crystal structures and cryo-EM structures of linear primer template dsDNA structures with magnesium and dNTPs and DNA polymerases are not informative about where 3rd strand of DNA is. The displaced strand is not the template and is not the primer.
Where is the displaced 3rd strand of DNA?
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Yes, that’s correct! In strand displacement synthesis, the primer strand is extended using the template strand, and each nucleotide incorporated displaces the third strand that was annealed initially to the template.
The third strand is a short, single-stranded DNA annealed to the template strand and acts as a stabilizing agent for the displaced strand . The displaced strand is the second strand of the double-stranded DNA being synthesized.
More at:
- Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB | Nucleic Acids Research | Oxford Academic (oup.com)
-D-loop - Wikipedia
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I need to dissolve sulphate to purify the nanozyme synthesized with magnesium sulphate
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thanks a million for your answer.
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  • Which is your favourite field of Medicine and why?
  • In which Medical field do you work?
  • What is your field of interest?
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Minha paixão é a psicanálise. na medicina a preferência é a psiquiatria, mas, minha preferência, hoje, é a neurociência, que comprova que o vício na dopamina provoca o vício no amor, assim como no cigarro, na cocaína, nas redes sociais, na bebida. ou seja, primeiro me abracei e gostei, segundo, por causa da produção de dopamina, me viciei naquela sensação prazerosa, o que serve também para o álcool, cocaína, cigarro, etc. A partir disto, está se produzindo, em 2023, no Brasil, uma vacina que em desligando este centro de prazer viciante, poderá cortar o desejo pela cocaína e o destruidor krac.
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Dear all,
I have been trying to knockdown my target protein using siRNA. The protein has several isoforms the effect of this knockdown has been showing up in the functional assay. However, I don't see the knockdown in the wesptern blot of the same test sample. Please suggest how to do an effective knockdown so that I can visualize that in the western blot as well.
Thank you in advance for your kind suggestions
Sincerely,
Prem
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You can definitely use multiple siRNAs. We use a pool of 4 as default regardless of isoforms.
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Hello,
We are trying to purify proteins using a secretion system but do not have TFF cartridges for our device. Does anyone know where we can purchase these? Or is there a better replacement? We want to downscale the volume from 3 L to 100-200 mL.
I've attached a picture for reference.
Thanks,
Thomas Newton
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PALL (now Cytiva) I think, used to sell TFF devices.
Tangential flow filtration | Cytiva (cytivalifesciences.com)
Another alternative could be Sartorius
TFF Systems For Ultrafiltration and Diafiltration | Sartorius
Hope it helps.
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I'm on the lookout for remote bioinformatics and computational biology opportunities where I can actively contribute to research projects. Compensation is not a priority for me; my main focus is to gain hands-on experience in these fields.
#biopython
#computational_biology
#bioinformatics
#biology
#R
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Avenues you can explore to find such opportunities:
1. Academic research institutions: Many universities and research institutions offer remote research positions or internships in bioinformatics and computational biology. Check their websites, job boards, and reach out to individual researchers or research groups who align with your interests.
2. Online job portals and platforms: Websites and platforms dedicated to remote work, such as LinkedIn, Indeed, and Upwork, often have listings for bioinformatics and computational biology projects. You can search for specific keywords like "remote bioinformatics," "computational biology," or "bioinformatics internships" to find relevant opportunities.
3. Open-source projects: Contributing to open-source bioinformatics projects can provide valuable hands-on experience. Explore bioinformatics software and libraries like Biopython, Bioconductor (for R), or other popular tools on platforms like GitHub. Contribute to their development, report issues, or collaborate with the community.
4. Online communities and forums: Engage with online communities and forums focused on bioinformatics and computational biology. These platforms, such as Bioinformatics Stack Exchange, BioStars, or community forums associated with specific software packages, often have job boards or project collaboration opportunities shared by researchers or organizations.
5. Networking: Attend virtual conferences, webinars, and workshops related to bioinformatics and computational biology. Connect with researchers, presenters, and fellow attendees to express your interest in remote research opportunities. Networking can often lead to potential collaborations or recommendations for available positions.
When searching for opportunities, it's important to tailor your search keywords to include relevant terms like "remote," "internship," "volunteer," or "project-based." Additionally, clearly communicate your enthusiasm, willingness to contribute, and desire for hands-on experience in your application materials or when reaching out to potential mentors or supervisors.
Hope it helps:credit AI.
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I intend to explore the integration of computational biology and artificial intelligence (AI) with laboratory and experimental work, encompassing animal models, cell culture, clinical trials, and molecular studies. As a clinical biochemistry student with a keen interest in AI, I believe this interdisciplinary approach holds immense potential for advancement and innovation.
However, I face the challenge of identifying relevant literature in this emerging field. I would greatly appreciate guidance on effective keywords and search strategies to navigate this landscape of research and achieve my research goals.
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I think there are several ways that they could be integrated. Each of those ways will have different ways to best approach this and a different literature review. For example what is the computational biology you are interested in - an DNA analysis is very different to molecular simulations or simulating a artificial hip. Biological laboratories can produce very different data a spead sheet is different to a image and the types of AI are different. It is common in computational science to want to compare simulation/computational results to experimental/observational results. I imagine this would be a good starting point for what you are interested in. In this case I think what you would want to do is use AI to segment or analyse images so that you have something to compare back to the simulation results. Can you give some examples of what you want to compare or research questions you want to be able to answer.
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My target protein is a membrane protein and I want to check its expression level in the test sample using western blot. Can anyone suggest which protein should be used for loading control, like we use beta-actin in cytosolic proteins. And is there antibody available for that ?
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When working with membrane proteins and performing Western blot analysis, it's essential to choose an appropriate loading control that is not affected by changes in membrane protein expression. Commonly used cytosolic loading controls, like beta-actin or GAPDH, may not be suitable in this case, as their expression levels can vary in response to changes in membrane protein expression.
Here are some options for loading controls when studying membrane proteins:
  1. Membrane Proteins as Loading Controls:In some cases, it may be possible to use another membrane protein as a loading control, especially if it is known to have stable expression across different conditions. Examples include various membrane transporters or receptors. However, finding a suitable membrane protein loading control depends on the specific context and characteristics of your experiment.
  2. Total Protein Staining:Use total protein staining methods, such as Ponceau S or Coomassie Blue, to visualize total protein on the membrane before antibody probing. This can serve as a general loading control, indicating the total amount of protein in each lane.
  3. Housekeeping Membrane Proteins:Identify housekeeping membrane proteins that are known to have relatively constant expression levels in the tissues or cells you are studying. Examples include Na^+/K^+ ATPase or V-type H^+-ATPase. However, it's essential to validate the stability of their expression in your experimental system.
  4. Use of Multiple Loading Controls:Consider using multiple loading controls to ensure the reliability of your results. For example, you could combine a membrane protein loading control with a total protein stain.
  5. Normalization to Total Protein Content:Normalize the intensity of your membrane protein of interest to the total protein content in each lane. This involves quantifying the intensity of your protein of interest and dividing it by the total protein intensity in the same lane.
As for the availability of antibodies, it depends on the specific protein you choose as the loading control. Antibodies against some common membrane proteins, such as Na^+/K^+ ATPase or V-type H^+-ATPase, are commercially available from reputable antibody suppliers. Ensure that the selected antibody recognizes the appropriate epitope and has been validated for Western blotting.
Before finalizing your loading control strategy, it's crucial to conduct preliminary experiments to validate the stability of expression of the chosen loading control(s) under your experimental conditions. Additionally, consider consulting the literature or seeking advice from researchers with expertise in the specific membrane protein and experimental system you are working with.
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I intend to explore the integration of computational biology and artificial intelligence (AI) with laboratory and experimental work, encompassing animal models, cell culture, clinical trials, and molecular studies. As a clinical biochemistry student with a keen interest in AI, I believe this interdisciplinary approach holds immense potential for advancement and innovation.
However, I face the challenge of identifying relevant literature in this emerging field. I would greatly appreciate guidance on effective keywords and search strategies to navigate this landscape of research and achieve my research goals.
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I think that Ross King (currently at Chalmers University of Technology) has a good number of publications that have made significant contributions to the subject of AI in science. You can look at some of his publications at one of the following links.
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I need to convert from micromoles per second per liter (µmol s⁻¹ L⁻¹) to millimoles per gram of dry cell weight per hour (mmol gDCW-1h-1)
Thanks!
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Wat bull. An "empirical formula" for a microorganism most of whose molecules are of profoundly greater molecular weight. But there's the "However..."
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Hello everyone! I am new to this platform, I am looking for this book: "Bioquimica del estres Oxidativo" (Diego Camps, 2010)
I would like to know if anyone has it in digital format to share it with me. Waiting for a reply. Thank you very much!
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I need to extract the liver from frozen Emerald rockcod and sequence its RNA. To keep the RNA stable and prevent degradation, I would like to avoid thawing the fish for the dissection. However, I haven't been able to find any methods that keep the fish frozen. Does anyone have any tips on how to best achieve this?
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Probably would need to work in a walk in freezer that is just below 0.
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If an active site mutant knocks out product formation at all excessive concentrations of substrate and at all excessive concentrations enzyme, but substrate binding affinity via anisotropy shows no difference in binding affinity for wildtype versus active site mutant enzyme, is kcat = 0? But if kcat = 0, then by the relationship of Michaelis constant (KM) to kcat then KM = KD.
How do you show to reviewers that the active site mutant is dead? If the wildtype mutant starts producing product in seconds, are you supposed to measure the reaction for the mutant for an hour at zero concentration of substrate and at 100fold excess substrate concentration of the K_M for the wildtype enzyme for the active site mutant, and show the time course?
Are there any publications that show a mutant enzyme is not just slow to produce product but is rather incapable of producing product but can still bind substrate? Examples would be greatly appreciated!
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Short answer - I would say yes. If an enzyme can't turn over products on any time scale, its turnover number would be 0.
I agree that if kcat = 0, then effectively your KM is just the ratio of kon and koff, that is, it is equal to the KD. If one was simply measuring binding of the substrate to the enzyme, I can definitely see that replacement of a residue critical to the mechanism, but not the binding and orientation of the substrate, would generate a variant that does not turn over product but still has a similar or identical KD for the substrate. This says nothing about the turnover number (or lack thereof).
How would I show a reviewer that a binding-competent enzyme is catalytically inactive? I think what you have described is going above and beyond. Extending a reaction for 60 min at 100 x KM to prove no activity exists feels a bit extreme. Even if the measured kcat is very tiny but not zero, it would be a moot point as this has absolutely no biological relevance.
If your wild-type enzyme has a kcat > 1 s-1 like you mention, I think it would be reasonable to measure the initial rates of i) the wild-type enzyme, ii) the catalytic mutant, and iii) a matched enzyme-free reaction using the same reaction conditions for all on whatever time scale is appropriate for your enzyme and its relative rate.
With enough replicates, you can use appropriate statistics to compare the grouped samples. Showing that there is no statistical difference in the rate of reaction between an experiment with no enzyme at all versus a catalytic mutant would be a strong argument that no activity exists, i.e. kcat must equal 0.
You will see in this paper we use an LC-MS assay to detect the presence/absence of product in active site replacements as justification for a dead enzyme, in addition to a colorimetric assay where we did the above to justify activity as "n.d." We did not measure affinity to the substrate but the binding site is > 15 angstroms away in this case.
Hope this helps!
ACA
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Hello all,
I hope everyone is doing well.
We are preparing buttermilk from standardized milk. In this regard we need to increase the pH of the buttermilk. We used sodium citrate (0.4% & 4%) and sorbitol (0.4%) to check any change in the pH. Unfortunately, there were no significant changes observed.
Please suggest any recommendation to increase pH using any natural or food-grade chemical compounds and any other alternative options.
Thank you in advance.
Keep smiling and Stay healthy.
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If this is for cooking, a quick recipe swap is to add vinegar (the acidity here is acetic acid) to regular milk to mimic the tangy flavor of buttermilk. It's a spoonful or two (about 15-30 ml) vinegar per 240 ml milk.
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I am reaching out to #researchers in the field of #Biochemistry, #Biophysics and #Bioinformatics, for collaborative partnership in scientific research. The researcher should be academic staff at the tertiary institutions in following listed countries:
#Afghanistan
#Angola
#Bangladesh
#Belarus
#Belize
#Benin
#Bhutan
#Burkina Faso
#Burma
#Burundi
#CaboVerde
#Cambodia
#Cameroon
#CentralAfricanRepublic
#Chad
#Comoros
#Congo
#CookIslands
#Cuba
#Democratic People's Republic of Korea
#Democratic Republic of the Congo
#Djibouti
#Dominica
#EquatorialGuinea
#Eritrea
#Eswatini
#Ethiopia
#Gambia
#Ghana
#Grenada
#Guinea
#Guinea-Bissau
#Guyana
#Haiti
#Iran
#IvoryCoast
#Kenya
#Kiribati
#Kyrgyzstan
#Lao People's Democratic Republic
#Lebanon
#Lesotho
#Liberia
#Madagascar
#Malawi
#Maldives
#Mali
#Marshall Islands
#Mauritania
#Micronesia (Federated States of)
#Mozambique
#Myanmar
#Nauru
#Nepal
#Nicaragua
#Niger
#Niue
#Palau
#PapuaNewGuinea
#Moldova (Republic of)
#Rwanda
#SaintHelena
#SaintLucia
#SaintVincent and the #Grenadines
#Samoa
#SaoTome and #Principe
#Senegal
#Sierra Leone
#SolomonIslands
#Somalia
#SouthSudan
#Sudan
#Suriname
#Syrian Arab Republic
#Tajikistan
#Timor-Leste
#Togo
#Tokelau
#Tonga
#Tuvalu
#Uganda
#Ukraine
#Tanzania (United Republic of)
#Vanuatu
#Yemen
#Zambia
#Zimbabwe
Interested researcher should kindly email to hezesapience@gmail.com with the subject: Research Collaboration from "your country".
Thanks.
Toluwase H. Fatoki
Visionary @ Heze-Sapience International, Nigeria.
Lecturer @ Department of Biochemistry, Federal University Oye-Ekiti, Nigeria.
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And why don’t you want any collaboration from Nigeria?
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Is there any system with small molecule binders and a short protein tag that is higher affinity than 6xHIS-NTA?
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V5 and Flag tag are the only ones I've seen other than His.
Einhauer A, Jungbauer A. The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J Biochem Biophys Methods. 2001 Oct 30;49(1-3):455-65. doi: 10.1016/s0165-022x(01)00213-5. PMID: 11694294.
Also consider the MBP that is engineered with a linker with a protease cut site. MBP sticks to the affinity column, and you use the protease to elute your protein of interest. Then of course the protease has to be purified out, so it should have an affinity tag or biotin.
Wikipedia has a long list.
Peptide tags
  • ALFA-tag, a de novo designed helical peptide tag (SRLEEELRRRLTE) for biochemical and microscopy applications. The tag is recognized by a repertoire of single-domain antibodies [5]
  • AviTag, a peptide allowing biotinylation by the enzyme BirA and so the protein can be isolated by streptavidin (GLNDIFEAQKIEWHE)
  • C-tag, a peptide that binds to a single-domain camelid antibody developed through phage display (EPEA)[6][7]
  • Calmodulin-tag, a peptide bound by the protein calmodulin (KRRWKKNFIAVSAANRFKKISSSGAL)
  • iCapTag™ (intein Capture Tag), peptide-based a self-removing tag controlled by pH change (MIKIATRKYLGKQNVYGIGVERDHNFALKNGFIAHN). Its patented component derived from Nostoc punctiforme (Npu) intein. This tag is used for protein purification of recombinant proteins and its fragments. It can be used in research labs and it is intended for large-scale purification during downstream manufacturing process as well. The iCapTag™-target protein complex can be expressed in a wide range of expression hosts (e.g. CHO and E.coli cells). It is not intended for fully expressed mAbs or membrane proteins[8][9][10]
  • polyglutamate tag, a peptide binding efficiently to anion-exchange resin such as Mono-Q (EEEEEE) [11]
  • polyarginine tag, a peptide binding efficiently to cation-exchange resin (from 5 to 9 consecutive R)
  • E-tag, a peptide recognized by an antibody (GAPVPYPDPLEPR)
  • FLAG-tag, a peptide recognized by an antibody (DYKDDDDK)[12]
  • HA-tag, a peptide from hemagglutinin recognized by an antibody (YPYDVPDYA)[13]
  • His-tag, 5-10 histidines bound by a nickel or cobalt chelate (HHHHHH)Gly-His-tags are N-terminal His-Tag variants (e.g. GHHHH, or GHHHHHH, or GSSHHHHHH) that still bind to immobilised metal cations but can also be activated via azidogluconoylation to enable click-chemistry applications[14]
  • Myc-tag, a peptide derived from c-myc recognized by an antibody (EQKLISEEDL)
  • NE-tag, an 18-amino-acid synthetic peptide (TKENPRSNQEESYDDNES) recognized by a monoclonal IgG1 antibody, which is useful in a wide spectrum of applications including Western blotting, ELISA, flow cytometry, immunocytochemistry, immunoprecipitation, and affinity purification of recombinant proteins [15]
  • Rho1D4-tag, refers to the last 9 amino acids of the intracellular C-terminus of bovine rhodopsin (TETSQVAPA). It is a very specific tag that can be used for purification of membrane proteins.
  • S-tag, a peptide derived from Ribonuclease A (KETAAAKFERQHMDS)
  • SBP-tag, a peptide which binds to streptavidin (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP)[16][17][self-published source?]
  • Softag 1, for mammalian expression (SLAELLNAGLGGS)
  • Softag 3, for prokaryotic expression (TQDPSRVG)
  • Spot-tag, a peptide recognized by a nanobody (PDRVRAVSHWSS) for immunoprecipitation, affinity purification, immunofluorescence and super resolution microscopy
  • Strep-tag, a peptide which binds to streptavidin or the modified streptavidin called streptactin (Strep-tag II: WSHPQFEK)[2]
  • T7-tag, an epitope tag derived from the T7 major capsid protein of the T7 gene (MASMTGGQQMG). Used in different immunoassays as well as affinity purification Mainly used [18]
  • TC tag, a tetracysteine tag that is recognized by FlAsH and ReAsH biarsenical compounds (CCPGCC)
  • Ty tag (EVHTNQDPLD)
  • V5 tag, a peptide recognized by an antibody (GKPIPNPLLGLDST)[19]
  • VSV-tag, a peptide recognized by an antibody (YTDIEMNRLGK)
  • Xpress tag (DLYDDDDK), a peptide recognized by an antibody
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Dear all, please suggest or provide a link for the provider or company who can design or synthesize shRNA plasmid (Single vector system) that will directly express into the mammalian cells to knockdown the protein expression for long term. Any suggestions will be highly appreciated.
Thank you
with kind regards
Prem
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Hi Prakash,
The Santa Cruz Biotechnology, Inc. (https://www.scbt.com/home) company offers almost all ready-made shRNA or siRNA for known protein expressed in mammalian cells. You can search for your interested protein in HOME page. Some of their shRNA and siRNA used in our current and previous study is of good characteristics. I think this may be useful to your work.
Regards.
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I am trying to express several proteins at the same time, but I want to use a different promoter and terminator for each one to avoid the possibility of recombination.
The promoters that are available to me are: TEF2, PGK1, CCW2, TDH3 and HHF2. The available terminators are: ENO1, SSA1, ADH1, PGK1 and ENO2.
Has anyone ever used these combinations of promoters and terminators? In your experience, which combinations work the best?
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Hi there,
These are strong constitutive promoters and terminators. Any combination should be OK... I have successfully expressed simultaneously 6 human proteins from genomic insertions using the following combinations: ProPGK1+terTDH1; ProTDH3+TerADH1; ProHHF2+TerSSA1; ProCCW12+TerENO1; ProTEF1+TerENO2; ProTEF2+TerPGK1. The most crucial point being to optimize sequences for expression in yeast.
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Hi
I'm purifying some mutants of the protein I study. The wild type protein exists as a monomer and is 28kDa.
I have two mutants (same protein, same number of amino acids but with 8 amino acid substitutions at defined positions), one of the mutants (mutant 1) analysed using size exclusion chromatography with multi-angle static light scattering (SEC-MALS) and its MW was shown to be 33kDa and has an oligomerization state of 1.2. The other mutant, mutant 2, also measured by SEC-MALS was 59kDa with an oligomerization state of 2.2.
For the wild type to measure the concentration I've just been using the MW (28kDa) and extinction coefficient (calculated by entering the sequence into online software ProtParam) and using a NanoDrop measuring absorbance at 280. This gives the concentration in mg/ml which I then convert to molar concentration.
For the mutants I want to measure their concentration the same way - measuring A280 on the NanoDrop using the mutants MW and extinction coefficient and calculating molarity from mg/ml. I'm not sure if this is an obvious/stupid question but what MW weight and extinction coefficient would you use for the mutants on the NanoDrop? E.g. For example mutant 2 molecular weight (MW) of the protein based on its amino acids (AA) composition is predicted to be 28kDa, but SEC-MALS shows it is 59kDa as the protein forms an oligomer.
My instant is to use 59kDa and the computed extinction coefficient predicted from the AA composition - is this correct?
Thanks in advance!
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Thanks for your reply very helpful - so if I’ve understood you properly - it is incorrect to use the SEC-MALS molecular weight when measuring UV 280 absorbance to determine protein concentration of the mutants as the extinction coefficient is based on the protein being denatured. Rather I should use the predicted Mw just based on the amino acid composition.
I agree the deviation from whole numbers points to an oligomer mixture. I want to add these recombinantly purified mutants to permeabilised cells to observe their localisation (they are fluorescent proteins) so knowing concentration accurately is important for these experiments.
So for example for mutant 2 whose Mw just based on amino acid composition is 28kDa - if A280 on the nanodrop gives a concentration of 10 mg/ml which would be 0.35mM, the concentration protein is 0.17mM? (as the oligomerizaiton state is 2 taken the closest integer value). I should say the ability of these mutants to form oligomers is because the mutations increase their aggregation propensities.
Am I correct in this? Is the above way accurate or would something like the Bradford assay be better?
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I apologize and excuse the owner of the post. I would like to invite you to read my ebook and discover why microorganisms are so fantastic. https://www.amazon.com.br/dp/B0CF1VKKK8
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PhD biochemistry candidate is required for the post of guest faculty. Interested candidates can contact me asap.
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PhD biochemistry from Iraq. From faculty of sciences /University of Kufa.
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I learned DNS assay to find out the quantity of maltose in the sample.
I realized the importance about the boiling water in this assay. But I got a several question after that.
I don't know the reason why do I have to cool-down the sample after heating up?
And also is ice a tool used to reduce cooling time, or is it used to cool quickly?
Please leave me a response about this problem.
Thank you for your help.
Joonseo_Cha The Student of Hallym University
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On heating the alkaline mixture of reducing sugar with DNS at elevated temperature (100°C) for 10 minutes, the reddish-orange color will develop, and at this point it will show a direct relationship with the concentration of the reducing sugar in the sample. The color developed is maximum at this time point (10 mins) which is standardized for this assay.
So, to stop the reaction immediately, the tube is dived in cold water. When the tube reaches room temperature, 300ul of the resulting reaction mixture is transferred to the well of a microtiter plate and the absorbance read at 540nm using microtiter plate reader.
So, when you use ice, it will cool down the sample quickly so as to stop the reaction in the tube.
Best.
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The size differ little bit from one species to another, yet they have one size range. Also, the size of them in their native form so they don't lose their colour while isolation.
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Uniprot is a good website for researching specific proteins. For example, you can type phycoerythrin and it will pull up all proteins with that name. Each organism will have a separate entry. Then you can look at data that has been generated by other researchers on that specific protein, including size, function, sequence, etc.
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I saw these on a same step of conversion of Malonyl CoA to Malonyl-ACP. Could anyone clarify this? I add that reference link below.
KEGG PATHWAY: Fatty acid biosynthesis - Chlorella variabilis (genome.jp)
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In FAS i.e., fatty acid synthesis the chemical reaction is, Malonyl CoA+ Acyl-carrier protein↔ CoA+Malonyl-[acyl carrier protein]. In fact, there are two substrates for the enzyme concerned. Concerned enzyme is a transferase. The enzyme very often called as S-malonyltransferase may be termed as FabD, i.e., Acyl-carrier-protein- S- malonyltransferase.
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I'd like to perform a SEC step prior to on-column refolding of a protein I am expressing/purifying but am worried about the extended time the protein will be in the presence of urea (and subsequent protein carbamylation) in the current refolding workflow I have.
Is there any concern of protein modification with a 4-6 hour denatured protein purification workflow using 6M GndHCl?
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I haven't heard of this being a problem.
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I am a second year PhD student at Wayne State University looking to screen a library of photosensitizers (transition metal complexes) for activity with ultrasound irradiation in cell cultures (likely MCF-7A cells). I would ideally like to set this up in a 96-well plate to screen multiple compounds but I am unsure of the set up as previous literature has not been as helpful as I would hope. The instrument I have is 3 MHz with a 3.5 cm diameter probe (Model-Sonic 103, Preamonex). I am unfamiliar with soundwaves so I will briefly discuss what I know so far from previous literature.
Overall, 0.3 W cm–2, 3.0 MHz seems to be a fairly common treatment condition when combining compounds with cell cultures.
This source clusters samples in a 2x2 square in the well plate and places the probe underneath the plate per cluster (see SI). Does a coupling agent need to be used and can this be done while keeping the plate sterile for continued incubation? Do the ultrasound waves pass through the plate to other clusters of wells (in a way that would significantly affect the other clusters)? Is there a way to evenly apply ultrasound stimulation evenly across the whole plate?
This source works with nanoparticles and a 96-well plate, but details of the set up are not given. Unless I'm missing something?
This source appears to place a sponge in degassed water on the head of the 35 mm probe. Could this be a valid option for the cluster of wells (2x2 square) mentioned above?
For the purpose of transfection, a 6-well plate was put into an ultrasound water bath.
Ideally, I would like to use the probe (as opposed to a water bath) to keep the instrumentation consistent between in vitro and in vivo studies. As I mentioned, I'm very unfamiliar with sonication/ultrasound and its physics. Any assistance/advice on how to make an accurate and precise high throughput set up is appreciated.
Thank you in advance!
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Setting up a high throughput system for ultrasound irradiation/sonication, especially for your specific application of screening photosensitizers in cell cultures, requires careful consideration of various factors. Given your current understanding and equipment, here are some steps and considerations to help you set up an accurate and precise high throughput system:
Coupling Agent and Sterility:
  • A coupling agent is often used to facilitate the transmission of ultrasound waves from the probe to the sample. It helps minimize air gaps that can impede wave propagation. Sterile coupling gels or fluids can be used to maintain aseptic conditions for continued incubation.
  • Plate Positioning:
  • Based on the literature you mentioned, positioning the probe underneath the plate in a 2x2 square cluster seems to be a common approach. This can help focus the ultrasound energy on specific wells. The use of a coupling agent between the probe and the plate is crucial for effective transmission.
  • Plate Material and Ultrasound Transmission:
  • The choice of plate material can affect the transmission of ultrasound waves. Opt for plates that are compatible with ultrasound transmission, such as plastic or materials with low acoustic impedance. Glass plates might attenuate the ultrasound energy.
  • Even Ultrasound Stimulation:
  • Achieving even ultrasound stimulation across the entire plate can be challenging. The setup you mentioned with a probe underneath each cluster can help localize the energy, but it might not provide uniformity across the entire plate. Experimentation and optimization are key to finding the best setup for your specific needs.
  • Impact on Adjacent Wells:
  • Ultrasound waves can interact with neighboring wells. While some energy might pass through to adjacent clusters, the effect would likely be attenuated. However, it's important to monitor this potential interaction, especially if you're aiming for precise and controlled results.
  • Probe Configuration:
  • Since you're using a probe (as opposed to a water bath), consider whether modifying the probe configuration, such as using an array of smaller probes or optimizing the positioning, could help achieve more even energy distribution.
  • Calibration and Dosimetry:
  • Accurate dosimetry is essential for consistent results. Calibrate your ultrasound system to ensure that the intended energy levels are being delivered to the samples. This calibration can involve measuring the intensity, power, and frequency of the ultrasound waves at different points within the plate.
  • Temperature Monitoring and Control:
  • Ultrasound irradiation can lead to localized heating. Monitor and control the temperature during experiments to ensure that any effects observed are due to sonication and not temperature-induced changes.
  • Safety Considerations:
  • Ultrasound can induce cavitation, which may affect cells and compounds differently. Carefully consider safety protocols and ensure that the chosen parameters do not cause harm to the cells or compromise the integrity of your experimental setup.
  • Collaboration and Consultation:
  • Collaborate with experts in the field of ultrasound and sonication. Consult with colleagues, mentors, or specialists who have experience in similar setups to gain insights and advice.
Remember that achieving an accurate and precise high throughput system requires iterative experimentation and optimization. As you become more familiar with ultrasound physics and its interaction with your samples, you'll be better equipped to fine-tune your setup for optimal results.
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How often do you get new salts? What salts do you get "fresh" most frequently? All help is greatly appreciated. Thank you to everyone who reads or responds, hope you all have a fantastic day.
I am a Graduate Student at the University of Wisconsin Milwaukee. Our most recent electrophysiology experiment was going fantastic for about 3 weeks. Suddenly, every slice was very poor quality, and every cell we patched onto died within about two minutes. Even if you don't have an Electrophysiology background, all advice and input is appreciated. It's been two weeks since this problem first arose. We didn't alter any procedures, everything has been held constant. We don't know what could be causing the sudden change in slice quality.
So far we have three theories: DDH20 filter needs replacing, our glassware has somehow become contaminated, or our salts have gone bad.
The DDH2O water resistance reads about 14.8MOhms. We changed the filter about a year ago. From what I have read, DDH2O should be between 14 and 18 MOhms to ensure slice quality, so we think our water is fine.
Our glassware washing procedure is 3 rinses of tap water, 3 rinses of deionized water, and 1 rinse of DDH2O. We don't know how else we can safely clean the glassware for slice preparation. But we don't think this is the issue.
Our main theory is the salts have gone bad. Our lab is on the 4th floor, and it is usually quite warm and humid on our floor during the summer. Most of opened salt bottles we use were first opened between 2 and 7 years ago. (for example, our bottle of sodium phosphate monobasic monohydrate and our potassium chloride were both opened 6 years ago. Our magnesium chloride and sodium chloride were both opened 2 years ago)
We think with repeated opening of the salt bottles causes debris/water from the atmosphere to leach into the salts, resulting in a change in our solutions and causing the cell death we have been observing. Is this a real possibility? How often do you get new salts? What salts do you get "fresh" most frequently?
We checked the pH, the pH of our solutions is within .1 of what it should be. All help is greatly appreciated. Thank you to everyone who reads or responds, hope you all have a fantastic day.
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These salts cost very little, and I would simply throw them out and buy new ones. If the issue goes away, great, If not, then you have narrowed down the options very quickly with little cost. You could also buy a bottle of sterile water to rule out some issue with your production system (I think you can have contaminants that do not affect the resistivity).
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I am currently making bacmid and consequent baculovirus using pDEST8, pFastBac and 438a vectors and recently switched to DH10EmBacY cells instead of DH10Bac due to the added YFP signal to monitor transfection, etc.
I was wondering if there is any reason for me to not use either of the two competent cells interchangeably to make bacmid DNA?
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DH10Bac and DH10EmBacY cells are both used for the generation of recombinant baculoviruses for protein expression in insect cells. However, they have different characteristics and uses.
DH10Bac cells are used in the Bac-to-Bac system developed by Thermo Fisher Scientific. They are used to generate recombinant bacmids containing your gene of interest in a baculovirus genome. These bacmids can then be transfected into insect cells to produce recombinant baculoviruses.
On the other hand, DH10EmBacY cells are part of the EMBacY system, an alternative system for generating recombinant baculoviruses. This system also involves creating recombinant bacmids, but the process and the specific cells used differ from the Bac-to-Bac system.
In general, you should follow the protocol and guidelines provided for each specific system and use the recommended cells for that system. Interchanging the cells may not produce the desired results, as each system has its own optimized conditions and requirements.
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I observed an interesting pattern that resembles a "flattening" of the near-UV region of the absorbance spectrum of the rat intestinal mucus in the streptozotocin-induced model of sporadic Alzheimer's disease. I'm studying qualitative and quantitative alterations in mucus as there seem to be some changes in the gut-brain axis, intestinal redox homeostasis, and cell turnover ( , ). I observed a quite dramatic change in the number and responsiveness of goblet cells so the observed spectral shift may be driven by a component of mucus. Another possibility may be a component of bile, a peptide or bilirubin/biliverdin, or even complexation between bilirubin/biliverdin and some other molecule (e.g. I found a similar pattern in a publication by Klinke et al. who reported flattening of the biliverdin near-UV spectrum upon binding to bacteriophytochrome? ). Any ideas about what I may be looking at?
Thanks!
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The report of UV-visible wavelength range in quantitative tissue spectroscopy has increased over last few years. The optical spectroscopy findings quantitatively in the UV-wavelength region might be a focus not only for study of malignancy but also other ailments too. I do suggest to go through the publication- Quincy Brown J, et al. Curr Opin Biotechnol 2009; 20:119-1311.
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Dear community,
I am planning on conducting experiments for which I need to obtain a plasma-free platelet suspension from an aliquote of a platelet concentrate. Do you now any methodology/protocol that allows for washing without extensive cell activation? I need the cells to be a "functional" as possible.
Yours sincerely,
Michael
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Platelet washing is very much needed not only for research purposes but also for preventing some transfusional reactions like anaphylaxis, fever etc. Human platelet-rich plasma(PRP) prepared from blood collected in tri-sodium citrate might face its aggregation owing to lower ionic calcium concentration. To get rid of this, isolation and washing of platelet is done with acid-citrate-dextrose solution and the washed platelet cells are suspended in a buffer solution containing 2mM calcium ion solution.
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I would like to add BG-azide to cells for SNAP tag pulldown however I don't know whether it is going to be cell permeable. My instinct is to day yes it will be but neither me nor the supplier know whether that is true. I thought I would ask if anyone has tried something similar, even though that is unlikely... I have attached the structures in case that is informative
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Thanks Wolfgang, I appreciate the detailed answer!
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In a patient with desminopathy (mutation Thr341Pro DES in the heterozygous state) with the progression of the disease, we note signs and symptoms that are also characteristic of botulism: bradycardia, arrhythmia, AV blockade, a significant decrease in the average duration of motor unit potentials according to electroneuromyography, paresis and paralysis of the striated muscles, decreased sweating, paresis of the gastrointestinal tract, dry eyes, dry mouth, symmetry of neurological symptoms, hoarseness, impaired visual acuity, doubling of objects occurs, progressive muscle weakness. These signs and symptoms are characteristic of botulism, only when a case of desminopathy is detected, they proceed slowly.
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Your analogy is very interesting, dear colleague.
Although the main cause of any form of myofibrillar myopathy is a violation of the structure of the protein components of sarcomeres caused by genetic mutations, why not assume that due to mutations, the sensitivity of the postsynaptic membrane of myofibrils in myofibrillar myopathy to acetylcholine may also be impaired.