Questions related to Biochemistry
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?
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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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.
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
How many grams of K2Cr2O7 to dissolve it in 1 liter Distilled water to obtain 50 ppm of Chromium? to become aqueous solution, Is there a specific equation to apply? Thanks
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.
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)
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?
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!
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.
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?
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?
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 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.
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
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.
I have protein with concentration of 0.6 mg/mL
The total volume of my protein is 4 mL.
Protein size is ~18 kDa
How can I convert my total protein concentration to micro molar?
There seems to be some consensus on the reaction mix for lactate dehydrogenase (LDH) activity assays, but the composition of the homogenization buffer (HB) differs wildly among studies. Maybe somebody can help me with the following three questions:
- Imidazole in HB: used in ~50% of studies in concentrations of 50-150 mM. Alternatively TRIS-HCl is used (50-75 mM). Almost all studies use imidazole in the reaction mix. Are there benefits to using imidazole in the HB instead of TRIS-HCl?
- Protease inhibitors (e.g., PMSF) in HB: used in few studies. Is this just for the workgroups that have more money to spend on chemicals?
- Some studies use KCN in the reaction mix without any explanation (earliest study that I could find was Sidell et al. 1987). Why?
Any recommendations and/or tips regarding LDH assays are greatly appreciated. Thanks!
Studies polled for this question:
Chippari-Gomes AR, Gomes LC, Lopes NP, Val AL, Almeida-Val VMF (2005) Comp Biochem Physiol B Biochem Mol Biol 141:347–355. https://doi.org/10.1016/j.cbpc.2005.04.006
Driedzic WR, Fonseca de Almeida-Val VM (1996) J. Exp. Zool. 274:327–333. https://doi.org/10.1002/(SICI)1097-010X(19960415)274:6<327:AID-JEZ1>3.0.CO;2-Q
Gerringer ME, Yancey PH, Tikhonova OV, Vavilov NE, Zgoda VG, Davydov DR (2020) FEBS J 287:5394–5410. https://doi.org/10.1111/febs.15317
Heinrichs-Caldas W, Almeida-Val VMF de (2021) Fish Physiol Biochem 47:1759–1775. https://doi.org/10.1007/s10695-021-01000-0
Martinez ML, Chapman LJ, Grady JM, Rees BB (2004) J Fish Biol 65:1056–1069
Moon TW, Mommsen TP (1987) J. Exp. Zool. 244:9–15. https://doi.org/10.1002/jez.1402440103
Sidell BD, Driedzic WR, Stowe DB, Johnston IA (1987) Physiol Zool 60:221–232. https://doi.org/10.1086/physzool.60.2.30158646
Singer TD, Ballantyne JS (1989) J. Exp. Zool. 251:355–360. https://doi.org/10.1002/jez.1402510312
I recently received invitations to contribute an article to the current issue of the Journal of Brilliant Engineering (BEN) and Chemistry and Biochemistry. Can anyone with experience or knowledge about these journals confirm their originality?
Hey all, I'm very interested in understanding more about the biochemistry behind all of the wash buffers that go into the streptavidin affinity purification protocol seen here (https://static1.squarespace.com/static/5617d7d8e4b09f2fdf34baa6/t/5fe2631ba775596e136098b6/1608672031257/Cho+NP+2020.pdf pg 3990, step #38).
I understand this is to help reduce non-specific interactors from being pulled down with the biotinylated proteins; however, I'm more curious to understand the necessity of the 2M Urea vs sodium carbonate vs KCl, etc....looks like previous iterations of this protocol have not used sodium carbonate and KCl buffers but I can't find substantiation for why they were added.
Thanks in advance!
I basically have normalized fluorescence data generated from a series of protein dilutions. I need to calculate K, K should be the same in all dilutions, thus the need for a global fitting approach. I have been using XLfit, but would like to move into a script format. Thanks in advance! Thus far, I found the package renz, but to my understanding, it does not accomodate for global fitting. Thanks in advance!
1-The conditions of the relationship between the pigment (Pyocyanin) with Gold (Au Nanoparticles)?
2-Dose the pigment (Pyocyanin) work as Reducing agent or not?
3-Dose the pigment (Pyocyanin) endures high temperatures or not?
One example would be MBP and MBP-74, an interaction which can be disrupted by maltose when it binds MBP.
Hansen Solubility Parameters full sets (dD, dP, dH and Ro) for
1) Cholesterol CAS#[57-88-5], and/or
2)Cholesterol Monohydrate with CAS#[5808-12-8] are needed.
Any or both experimental or calculated in HSPiP values are interesting.
Old published values (20.4, 2.8, 9.4 and 12.6) are off interest.
I am currently working on Co-IP of NRIP and MyoD by overexpressing both proteins in HEK293T cell. The question is I cannot get a specific band of MyoD. For my input, I think the protein level is too low to be detected so I will increase the protein amount to run a western blot. However, several bands appear when detected using MyoD antibody after co-IP. The band most likely MyoD is pointed with a blue arrow. Some of my colleagues say it may be endogenous MyoD or protein with similar conformation. Does anyone face a similar situation when working with this protein? or any reference paper suggested for studying this protein. Thank you.
Can alpha glucosidase and alpha amylase work at room temperature and at 37*C? If so why? The specifications for these enzymes are that they work at 37*C. But number of papers have modified their protocols 25*C. How can it work at both room temperature and at 37*C?
I'm sure this question has been asked a lot but the protein I am purifying is not as clean as I would like and all the potential solutions I have read about have not worked for me.
I am purifying a protein that forms inclusion bodies. The construct is 14x His tag - Tev cleavage - protein of interest. I use BL21 Star (DE3) cells and TB media for growing the bacteria and induce at OD 0.6 for 4 hours at 30 degrees.The purification protocol is based on a previously published paper. Following sonication, lysis buffer and washing the pellet the protein is extracted from the inclusion bodies using 6M Guanidine Hydrochloride, applied o/n at room temperature to a Ni NTA agarose column. It is then eluted in 4M Guanidine. I then pass it through a RP-HPLC. As you can see by the attached image although I am getting a high amount of protein it has a large smear which I am not sure if it is contaminants or degredation. I overloading with sample when trying to judge purity but I think it's better to get an accurate representation of what’s going on rather than kid myself I am working with a pure sample.
The HPLC makes no difference so I think optimisation on the Ni-NTA purification is needed but nothing thus far has worked, I have tested different induction temperatures, leaving it on the Ni column at both 4 degrees and for a few hours (rather than O/N at RT like the protocol says), protease inhibitors and washes/step-wise elutions with different concentrations of imidazole (as in attached file) - yet none of this has worked. I've even thought about making the His tag smaller (14x seems quite large, but I cant see how this would impact purity other than needing more imidazole for elution).
If anyone has comments on the purity and has any tips that I have not tried it would be greatly appreciated!
In speculating about plant migration from the oceans to land, I wondered if any plant species went back into the ocean. Since that is unlikely, this question arose to explain why.
In a study of plant genetics, this area of plant diversification is likely amazing, because of how the gene sequences are accessed and how complexity developed as new genes and biological systems.
We are doing a research on Biofilm formation of Bacteria, for knowing each isolate strong, moderate or weak, by calculations tables of Standard deviation, Variance and Cutoff (Ct) etc… and with the help of Microsoft Excel but the results in the program differ from the hand written and don’t know the best way to calculate and compare the results, any help will be very appreciated, Thank you
Bio-sensor development demands a variety of fields in cooperation. However, this breakthrough would not result in the case of doing research with a single attitude. I am calling researchers from biology or biochemistry, genetics, or related science to come into the discussion. Let me know if somebody has any knowledge in this field.
#Bio-sensors #Biochemistry #Genetics
My aim is to detect nascent protein synthesis by click-chemistry of L-homopropargyl-glycine-labelled proteins with biotin azide and detection by western blotting with fluorescently-conjugated streptavidin. So far, I have failed to detect labelling above what looks like background biotinylation. I'm using biotin azide from Biotium (Cat# 92167), but I've noticed there different biotin azide molecules with different molecular weights. I was wondering if there were differences in their applicability for copper-catalysed click-chemistry. Any help with troubleshooting this would be appreciated. Cheers!
Aside from inhibiting protease activity (not effectively working), I read some papers about doing modifications to the antibodies:
using unnatural amino Acids
But, are there better options, more commercially applicable?
Please spread the word: Folding at Home (https://foldingathome.org/) is an extremely powerful supercomputer composed of thousands of home computers around the world. It tries to simulate protein folding to Fight diseases. We can increase its power even further by simply running its small program on our computers and donating the spare (already unused and wasted) capacity of our computers to their supercomputation.
After all, a great part of our work (which is surfing the web, writing texts and stuff, communicating, etc.) never needs more than a tiny percent of the huge capacity of our modern CPUs and GPUs. So it would be very helpful if we could donate the rest of their capacity [that is currently going to waste] to such "distributed supercomputer" projects and help find cures for diseases.
The program runs at a very low priority in the background and uses some of the capacity of our computers. By default, it is set to use the least amount of EXCESS (already wasted) computational power. It is very easy to use. But if someone is interested in tweaking it, it can be configured too via both simple and advanced modes. For example, the program can be set to run only when the computer is idle (as the default mode) or even while working. It can be configured to work intensively or very mildly (as the default mode). The CPU or GPU can each be disabled or set to work only when the operating system is idle, independent of the other.
Please spread the word; for example, start by sharing this very post with your contacts.
Also give them feedback and suggestions to improve their software. Or directly contribute to their project.
Folding at Home: https://foldingathome.org/
Folding at Home's Forum: https://foldingforum.org/index.php
Folding at Home's GitHub: https://github.com/FoldingAtHome
Additionally, see other distributed supercomputers used for fighting disease:
Rosetta at Home: https://boinc.bakerlab.org/
When staining with hematoxylin and eosin of a muscle biopsy from a patient with T341P desminopathy, we observe accumulations of inclusions similar to nuclei (arrows in figures 1 and 2, x280). And outside of these accumulations - adipose tissue, which used to be muscle tissue. There are no such massive accumulations of inclusions in adjacent muscle fibers. We assume that clusters of inclusions are not nuclei? Figure 2 is the inverted figure 1.
For the protein and ligand preparation process what kind of structure should ı choose and what is the reason of that
I made several reactions with 3,5-Dinitrobenzoyl chloride. When I looked at similar reactions for this substance, no heat was ever given in the reaction (even though heat increases the yield).
The interesting thing is that this substance turns black as soon as the heat is given to the reaction environment. Due to the fact that, theoretically, the polymerization of this material is impossible. Do you know the reason why they do not give heat to this material?
I need to find a mutant allele of my protein that is defective for homo-oligomerization, but can still bind a separate protein (heterodimer formation).
I think I will do alanine scanning mutagenesis of my protein.
However, my assays are very low throughput and I must assay each mutant protein individually. I simply cannot assay every single mutant allele in the time that I have.
I do not have any structural information. And Alphafold cannot solve the structure.
I am considering:
1. Mutate 3 residues at once. Is this too much? Will I simply destabilize my protein?
2. Only mutate charged residues, as oligomerization fequently relies on charged residues from my research. Does this seem incorrect?
Or is there better mutagenesis approach than alanine scanning?
I am doing my thesis for the last season of college and ı need to dock to ligand and protein for better binding affinity ı decided to combine the two ligand for moluecular docking so how can I combine the 2 ligand into 1 receptor molecular docking.
Usually, plant phenols are best extracted with solvents like methanol and ethanol. But I would like to know if the extraction is carried out with water, how much usually is the efficiency of that extraction??
I project to measure levels of total phenolic compounds in peppers following the method of the Folin-Ciocalteu's reagent, and I am going to calculate them by reading absorbance using gallic acid as a standard.
Nevertheless, I am confused about what wavelenght should be setted: while some authors read absorbance at 765 nm (Dogan et al., 2018; Kupina et al., 2018); others read it at 760 nm (Ghasemnezhad et al., 2011); 750 nm (Toledo-Martín et al., 2015; Lwin et al., 2022) or even 725 nm (Vega-Gálvez et al., 2009).
I would be grateful if someone could give me some light about this.
Thanks to everyone in advance.
Dogan, A.; et al. (2016). Comparison of pesticide-free and conventional production systems on postharvest quality and nutritional parameters of peppers in different storage conditions. Scientia Horticulturae 207: 104-116.
Ghasemnezhad, M.; et al. (2011). Variation in phenolic compounds, ascorbic acid and antioxidant activity of five coloured bell pepper (Capsicum annum) fruits at two different harvest times. Journal of Functional Foods 3: 44-49.
Kupina, S.; et al. (2018). Determination of total phenolic content using the Foling-C assay: Single-Laboratory validation, First Action 2017.13. Journal of AOAC International 101 (5): 1466-1472.
Lwin, H.P.; et al. (2022). Perforated modified atmosphere packaging differentially affects the fruit quality attributes and targeted major metabolites in bell pepper cultivars stored at ambient temperature. Scientia Horticulturae 301: 111131.
Toledo-Martín, E.M; et al. (2015). Application of visible/near-infrared reflectance spectroscopy for predicting internal and external quality in pepper. Journal of the Science of Food and Agriculture 96: 3114-3125.
Vega-Gálvez, A.; et al. (2009). Effect of air-drying temperature on physico-chemical properties, antioxidant capacity, colour and total phenolic content of red pepper (Capsicum annuum, L. var. Hungarian). Food Chemistry 117: 647-653.
In the following article's ESI, it seems that in situ decomposition of 2,5-Dihydroxy-1,4-benzoquinone (DHBQ) lead to the formation of oxalate under hydrothermal conditions.
Can someone explain the mechanism/pathway behind it?
Ref.: Science 374, 1464 (2021)
Please correct me if I am wrong by any chance.
Thanks in advance.
I am trying to use a technique used by previous graduate students that involves growing yeast in SC media -Uracil buffered to pH 6.75. All the literature published from our lab states that the growth media was buffered to pH 6.75 with 100 mM HEPES.
My problem is that I have tried multiple times to buffer the solution and have not gotten a stable pH through the growth cycle.
Initially, I prepared 1M HEPES buffer pH 6.75 and diluted it 10X to reach 1X media concentration, however upon dilution to 1x, the pH drops significantly. I then prepared the 1X media with the 100 mM HEPES, and adjusted the pH with NaOH and KOH (two separate attempts), however the pH was not stable through during growth and acidified significantly. I then buffered the 1X media with a pH 6.5 and pH 7.5 100 mM HEPES buffer, and the same issues persisted.
Does anyone have any insight into what I an missing here?
Hello everyone. I need a little help here.
can we use essential oils to reduce chlorinous odor in a hypochlorous acid solution without decreasing free chlorine or pH value? any toughts and propositions are much appreciated
i am a medical student, however i would like to learn molecular biology to the extent that makes me comfortable with putting a hypothesis, a theory or possible treatment and testing this with the help of a specialized research team and as such, i want to reach to the point where i fully understand modern molecular biology and specially the LAB TECHNIQUES. What is the process that i should follow in order to achieve this.
keeping in mind that i have near full access to springerlink, RSC and online wiley libraries
I am trying to express and purify nanobodies from a nanobody library in E. coli. I am using a pBAD vector to which I added the pelB sequence for periplasmic localization. The nanobody is fused to YFP with a His tag.
I induce the expression with arabinose and I can see on an SDS-PAGE gel that the nanobody is being expressed (although the expression seems to be lower than for some of the other proteins that I am expressing using the same vector), but I am having trouble with the extraction and purification steps.
I have tried to extract the nanobodies using lysozyme with PMSF following a protocol that usually works for me and I have also tried the osmotic shock protocol (
I do not have any experience with nanobodies so maybe there is an important step in the protocol that I am missing or not doing properly. I would appreciate tips or good protocols for expressing and extracting nanobodies.
I got results after performing an enzyme kinetics with 4 different substrate the kcat of B and D is respectively two times lower than A and C. However, I am seeing the higher Km of A i.e 174 nM as compared to B i.e 54 nM. Can anyone please suggest me how to rationalize this result ? The Kd value however, is always doubled for A and C when compared to B and D respectively. Below are the data attached for your reference. How could it be explained the research paper ?
I have preformed a dehyrogenase assay on an arid Negev soil. the calibration curve highest values were about 0.5 but the values for some of my samples were even 2 or 3 times that based on the protocol Alef, K. "Dehydrogenase activity." Methods in applied soil microbiology and biochemistry. London: Academic Press, 1995. 228-231.
also the results were much higher on the FDA protocol based on:
Adam, G. and H. Duncan. 2001. Development of a sensitive and rapid method for the
measurement of total microbial activity using fluorescein diacetate (FDA) in a range of
soils. Soil Biol. Biochem. 33:943-951.
the curve values were 0.05 to 0.1 and the values were 0.2 -0.7. is this normal for soils? some of them were poluted 5 years ago
It requires about 5.3 kcal/mol (or 8 kBT) of energy to break one phoshodiester bond of DNA. How do these enzymes cut the DNA only by using thermal energy and not ATP? I am only considering the ATP-independent restriction enzymes (Type II). How do these enzymes manage to generate the necessary energy? I couldn't find the exact mechanism with energetics of restriction enzymes cleaving DNA. Please provide me any relevant references.
A little question about PyMOL: Assuming that I know a coordinate, and now I want to see which atom this coordinate belongs to in the structure, what select command should I use? Namely, "select selection_name," + what?
I didn't find any materials on this.
Also, attached is the selection algebra of PyMOL (https://pymolwiki.org/index.php/Selection_Algebra).
Does anyone know why they say x<12.3, etc.?
Thank you very much!
3 dpf larvae of zebrafish will be used to evaluate the body burden concentration of a compound at different period of time.
The question is: Determining hexokinase activity in rat brain where 10 µl of a 10% (100 g/L) brain homogenate (containing hexokinase) is mixed with 1990 µL of reaction medium (containing the substrate glucose and the coenzymes ATP and NADP+ and the auxiliary enzyme glucose-6-phosphate dehydrogenase). The reaction mixture is placed in a cuvette (light path = 1 cm) in a spectrophotometer at 37°C, where the hexokinase reaction can be seen as an increase in absorbance at 340 nm. The reaction proceeds at a constant rate during the measurement period, and during 10 min a total increase in the light absorption at 340 nm of 0.10 is recorded. The absorption coefficient of NADPH = 6300 x M-1 x cm-1
I need to find the enzyme activity of hexokinase in µmol/min/g. I don't understand how I need to interpret the first line (10 µL of a 10 % (100 g/L) brain homogenate) - and how should I use these numbers?
Thanks in advance!
A patient with desminopathy survived Covid-19 six months ago without pneumonia, but with a temporary loss of smell and taste. After Covid-19, we note an accelerated progression of desminopathy, penetration accelerates, new muscles are quickly involved in the pathological process, muscle mass decreases, and heart function worsens. Perhaps the infection or its consequences are somehow connected with the mechanism of progression of desminopathy?
I was wondering if anyone could point me to any references that show that with increasing oligo length, there is a significant reduction in ssDNA generation efficiency. Im specifically curious at what point heating to separate dsDNA becomes ineffective and some sort of legitimate physical experiment to quantify this reduction with increasing DNA length.
I'm going to be labeling different size thiol-activated polyethylene (PEG) molecules with Atto488-Maleimide. I'm wondering what is the best way to accurately measure the final concentration of the PEGs following labeling and removal of free dye.
Colourmetric assays like BCA and Bradford would not work right for PEGs, neither would measuring A280 on a Nanodrop - so the only thing I can think of is either:
a) measuring the UV absorbance of the atto488 dye and using dye concentration as a proxy for the amount of labeled PEG (assuming I have high labeling efficiency).
b) Lyophilize the final product and just go by dry weight
Has anyone got any advice as to what the best way would be/any other suggestions?
I need to characterize two extra cellular enzyme produced by bacteria . For that process the purification of enzyme step is necessary before the characterization process? otherwise can i use the crude enzyme extract (cell free Supernatant) for the characterization process?
Kindly discuss your ideas and viewpoints on the origin of life and the RNA world hypothesis.
What are the contradictory views on why researchers are still unsure about the origin of life through RNA or such analogous molecular intermediate pre-cursors preceding its existence?
"The general notion of an “RNA World” is that, in the early development of life on the Earth, genetic continuity was assured by the replication of RNA and genetically encoded proteins were not involved as catalysts. There is now strong evidence indicating that an RNA World did indeed exist before DNA- and protein-based life. However, arguments regarding whether life on Earth began with RNA are more tenuous. It might be imagined that all of the components of RNA were available in some prebiotic pool and that these components assembled into replicating, evolving polynucleotides without the prior existence of any evolved macromolecules. A thorough consideration of this “RNA-first” view of the origin of life must reconcile concerns regarding the intractable mixtures that are obtained in experiments designed to simulate the chemistry of the primitive Earth. Perhaps these concerns will eventually be resolved, and recent experimental findings provide some reason for optimism. However, the problem of the origin of the RNA World is far from being solved, and it is fruitful to consider the alternative possibility that RNA was preceded by some other replicating, evolving molecule, just as DNA and proteins were preceded by RNA." - Robertson and Joyce
[This is as per the explanation by Michael P Robertson and Gerald F Joyce in the article: "The origins of the RNA world." published in the Cold Spring Harb. Perspect. Biol. 4, a003608 (2012).]
The scientific community must resolve this contradicting conjecture through rational discussion and debate backed by strong experimental evidence on what must be the pre-cursor molecule to the Origin of Life if it is not RNA!