Questions related to Analytical Biochemistry
if gene expression is low in specific diseases based on previous research,
But when I measured the protein expressed by the same gene in blood of same disease by ELISA I found high serum level?
what is the explanation for this difference between gene expression and its protein concentration in blood?
Say you are measuring the concentration of metabolite of interest (MOI) using fluorescence detection. The MOI concentration the one and only sample is unknown, say, u mg/L.
By adding extra MOI at defined concentrations to the same sample, you have a series of fluorescence readings as follow:
Concentrations -- Fluorescence readings
n mg/L -- Reading 1
(n + 15) mg/L -- Reading 2
(n + 30) mg/L -- Reading 3
(n + 60) mg/L -- Reading 4
(n + 120) mg/L -- Reading 5
As MOI concentration increases, fluorescence increases.
What would you do to obtain the value of n?
I have purified crude glycerol which is come from as a byproduct of transesterification. I need to know, the purity of the sample after purification. What methods can I apply to get the glycerol purity?
I would like to use 100% methanol (-20 degree C) for fixing monolayer cell culture for ICC-type of procedures. Is there any requirement on the grade of methanol to be used? E.g. Sigma has this (Cat. No. 494437) methanol "Suitable for protein sequencing, BioReagent" and this (Cat. No. M1775) listed under "fixatives", while we have analytical grade methanol (for preparing Western blot transfer buffer) in the lab. Can someone advice which grade of methanol should I use? Many thanks!
Dear all, I have extracted lemongrass essential oil by Soxhlet extraction using hexane and methanol as the solvent. The problem is the waxy fraction that solidified in room temperature. Any suggestions? I will be using GC to determine the essential oil concentration.
I am taking an enzymatic solution of uricase and uric acid in presence of TMB and a catalyst for colorimetric sensing. The enzymatic solution is prepared by taking 3mg in 3ul Borate buffer of pH8.5. I was expecting to see a color change due to the formation of peroxide which should break down in presence of catalyst and TMB, and should give blue color. However I do not see any color change.
Any help is much appreciated!
As seen in the picture, the peak appeared in several retention times which is very weird (it has no fixed retention time) !!
Also, it appearedis later at 7 min and disappeard from 5 min !!
I analyzed the internal standard "2-methyl-L-cysteine hydrochloride" using HPLC and the chromatogram shows three peaks instead of one which is weird!!
I repeated the preparation process two times to check if there is a contamination problem but the chromatogram still showing three peaks. So what do you suggest?! Is there a possibility that the internal standard is converting to other compounds?!
I want to detect TCA cycle intermediates, pyruvate, glyoxylic acid with GC MS, I have MTBSTFA, after derivatization, pyruvate, oxaloacete and glyoxylic acid cannot be detected, does anyone know how to quantify those metabolites with one method? I want to use GC MS bot LC MS
Hope you are doing well.
I'd kindly like to ask if anyone may have any available internships/apprenticeships with regards to the field of research. I am a volunteer with IAESTE Malta and am currently in my second year completing a Bachelors of Science (Honours) with Chemical Technology where I place as one of the highest students in my class. I am looking for a 1-3 month period that focus' on spectroscopic technics or anything mycology and/or chemistry related.
I am asking here since I would like to have an experience outside of my country that would be both educational and enjoyable.
Anyone that may know of any offer and/or is offering one please leave a comment or email me through my student email: firstname.lastname@example.org.
Thank you for your time and considerations.
Thanks for your attention.
Few days ago, I detected the plant Tyrosineammonia-lyase (TAL) ,plant cinnamyl alcohol dehydrogenase (CAD), plant 4-coumarate:CoA ligase(4 CL) and plant L-Phenylalanine ammonla-lyase (PAL) by enzyme-linked immunoassay (ELISA) kits. And the results were expressed by density (U/L). So, whether these results could be represented as enzyme activity?
Imagine, you have no problem choosing one of the following disciplines.
Which of these disciplines do you choose? Be sure to state the reason for your choice.
Thanks a lot
Am in Peptide / Protein Research, may i know what useful analytical information we are getting from 13C & 1H NMR spectroscopy analysis?
Somebody getting NMR for smaller proteins, any useful information we will get it from this?
I have a difficult to dissolve drug, previously I used DMSO to dissolve it but I want to avoid the toxic effect of DMSO, so I am planning to dissolve it in methyl-cellulose and use tween 80 to emulsify it. Can I prepare it and use it within a week or should I prepare it everyday and use it freshly.
Thanks a lot,
I am trying to run my cyanobacteria extracts in GCMS, but, the GCMS lab-solutions software is indicating that the MS communication hardware is not connected also showing code 0D80. What could be the problem and how can I overcome it?
Three rare codons code for arginine out of them CGA arginine codon coded by which rosetta strain?
I am interested only rosetta strain which codes CGA rare codon.
I am doing a chemical desorption of phenol from granular activated carbon by organic solvents, and I want to know if is it possible to detect the quantity of phenol in solvent directly by using spectrophotometer at 270 nm wavelength and without using the method of 4-aminoantipyrine
I am wondering if the N-terminal methionine excision, which is common PTM, can be followed by the excision of the second residue such as Alanine?
I have analyzed different proteins from different projects and this double excision is visible.
I am running catecholamine standards in HPLC using UV/FLD detector. I am getting a constant peak at fix RT (retention time @ 3.5 min, image enclosed) every time, even if I run another standard or buffer (acetate buffer) or inject methanol or acetonitrile. I have washed the column, needle but still getting the peak. How can I correct it? Please advice
Please suggest a protocol for Urease assay by Nessler's method. The difficulty which we are facing is precipitation and non gradient colour at the end of reaction.
I have mostly done computational works and the transition to experimental work is slightly demotivating as I am stuck at each stage starting from whether to wear gloves for certain things to why my experiments are not reproducible!
At this stage, I am seeking an answer to how can I weigh my peptide correctly to make sure I am getting the same concentration as I planned?
For example, I want to get 1mM (just an example) concentration for my peptide and I calculated the required mass to be 0.7134 mg. Now a few questions which bother me are:
1. Since the weighing balance can only allow two decimal places, so should I round it off to 0.71 (because 3 is less than 5) or should I round it off to 0.72 (because it is likely that I will lose some peptides anyway while weighing!)
2. Even though I know theoretically the concentration of my solution, should I still measure maybe using nanodrop or some other way? (and how accurate it will be if no aromatic aa)
3. Is there anything else I should be taking care of here?
Also, if there is any authentic website or paper to read about this basics, please recommend.
Thank you all in advance.
Intermediates are very important and often found in protein unfolding or foldingbpath. Sometimes, the intermediates are characterized as molten globule states (native like secondary structure and partially perturbed tertiary structure), and pre molten globule states (probably intermediate state between molten globule and unfolded state). I have seen report to use the term highly ordered molten globule description for a-lactalbumin.
My question is if there is any attempt to categorise the intermediate states observed in the protein unfolding?
Secondly, I am unclear of the definitions of this molten globule or pre molten globule state.
I'm working on gelatin modification. To do this, the amine group was protected by BOC and the carboxylic group was activated by EDAC and NHS to form a stable amide bonds with another amino-content small molecule.
Now I need to remove the protecting group. As in the reference, they use the conc. HCl (0.5 % v/v to sample) at 4 degree Celsius to deprotect the BOC. But this spend about 2 weeks to reach the full deprotection.
I hope to fasten this process, I have found some papers such as using conc. HCl (0. 01% v/v) at 40 degree celsius take about 16 hours and HCl (4M)/dioxane at 0 degree celsius take around 30 mins for deprotection. Will these methods also break the amide bond of gelatin and the additional group that I'm grafting to the backbone? Anyone has experienced in this kind of reaction, please help !!!!
Potassium Sorbate added to cheese with low pH due to the presence of lactic acid; does lactic acid act the same as HCL to convert Potassium Sorbate to Sorbic.
Also, potassium Sorbate added to brine ( saturated water with salt 10% at 4 C degrees)
How you think these conditions will affect the equilibrium between potassium Sorbate to Sorbic acid.
The problem in food industry labs they rely on outside labs to test and analyze samples for all out of ordinary tests and I've been trying to find a lab to analyze Potassium Sorbate separate from Sorbic acid but they only test Sorbate by HPLC which doesn't give an idea how much of each component is in that sample.
We have facilitated with Shimadzhu 8040 LCMS. Actually I'm searching for a platform that perform untargeted metabolomics of saliva as well as cell lines. The untargeted metabolomics mainly focusing upon primary metabolites of human origin. I have already search for many research articles and journals for the shimadzu LCMS methodology for the untargeted metabolomics. However I cannot find out a proper method for the same. My questions are :- 1. weather this instrument (Shimadzu LCMS-8040 Triple Quadrupole Liquid Chromatograph Mass Spectrometer ) is well suited for the untargeted metabolomics or whole metabolomic profiling? Please advise some research articles or proper methodologies if available. 2. I also facing the problem on data analysis part of the shimadzu's LCMS output file format, that is data analysis software or any raw data converter to the universal formats like .netCDF or MZXML?
I want to set up an assay to estimate the level of beta oxidation in parasitic cells, I saw some radioactivity based assays available for the same but I am interested in calorimetry based or luminescence based assays. It could be kit based assay also.
Our lab is trying to look at the differences in metabolic profiles of control and HD (Huntington's disease) patients.
We have a few fundamental questions on how we should quantitate the endogenous metabolites using HPLC- ECD (Electrochemical detector- a non-specific detector like UV).
After reading some papers about the same question, I found that background subtraction and standard addition methods may not work for us, as they could be tedious or may not allow good sensitivity. However, I think the surrogate matrix approach could be our best bet because it may be more representative than quantitating from standards prepared in the solvent.
a) So, what can be a good surrogate matrix for human plasma? Is 5% BSA in PBS, a good one to quantitate metabolites in tryptophan and tyrosine pathways? I have read that in order to use a matrix as a surrogate matrix, recoveries must be similar in plasma and the surrogate matrix. In order to calculate the recoveries, should I make 3 sets of calibration curves, one in the solvent, one in plasma, and the other in 5% BSA in PBS? and calculate the recoveries in plasma and 5% BSA in PBS with respect to the calibration curve prepared in the solvent.
b) We also run a pooled sample (made from all patient samples) in every batch to check instrumentation effects. Even after preparing a calibration curve in a surrogate matrix, is it still necessary to normalize with the average pooled sample's value from all batches?
c) My plan is to have a batch with this template. The idea is to quantitate samples from the calibration curve made in the surrogate matrix and check for quality control using recoveries of spiked pool samples. Would you change this order?
Equilibration injections (2)
Calibration standards made in 5% BSA in PBS (low to high) (7)
Spiked pooled samples (3 levels and 3 respective controls with the same spike volume)
We were wondering if any of you had the same questions and if you could help us out? Please let me know if there are any articles or books that may help answer these questions.
Thank you very much for reading.
is there any method to quantify the concentration of xylose (xylose only, not in mixture form) other than using HPLC. for example, detection by using any assay, o uv-vis maybe?
We have an old Packard SpectraCount in the lab and we need a copy of the software to run it. Can anyone help out?
I thank you in advance!
I have read in some papers that nano liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is more sensitive than conventional LC-MS/MS. But they never really mentioned why. I'm wondering how nano flowrate can achieve higher sensitivity.
I am determining drug recovery of liposomes, before calculation of encapsulation efficiency, against a calibration curve using UVvis spec. I would like some advice on whether this is a correct way of doing it.
I intend on dissolving 1ml of my empty liposome suspension with 3ml ACN/Methanol (2:1) and using my empty dissolved liposome as the blank. So we have a liposome suspension in a 4-fold dilution of solvents. Can I simply put this in the spec and blank it, then take 1ml of my loaded lipsome suspension, add the 3ml solvents and dilute when necessary until absorbance reads <1?
My real question is I am wondering if the difference in dilution of my sample with the solvents for the blank versus the loaded liposome will negative affect my results and cause errors as a result of difference in absorptivity?
Hi dear researchers.
I have a mixture of nanoparticles and hemin and buffers, …
It was centrifuged and washed several times.
Is there any way to calculate the residual hemin concentration?
( There are not any biological samples)
Dear to whom it may concern,
I would like to ask people who are interested in univariate analysis in metabolomics. Now, I am proceeding my metabolomics data using univariare analysis, namely p-values and FDR-adjusted p-values.
However, as far as I know, the calculation of a p-value for each feature depends on two factors: (a) distribution of each feature and (b) variance of each feature between case and control group. To be more specific, the first step is that we need to apply a statistical tool (I do not know which tool can help me to check this issue) to check whether one examined feature is normally distributed in both these groups or in only one of them, and of course, there are two scenarios as follows:
1. If this feature is normally distributed in both these group, we proceed to use F-test as a parametric test to check whether the variance of this feature in both these groups is equal or unequal. If it is equal, we can do a t-test assuming equal variance, otherwise, a t-test with unequal variance must be taken into account.
2. If not, a non-parametric test will be applied to obtain a p-value for this feature. In this case, may you please show me which tests are considered as non-parametric tests?
I am unsure that what I mention above is right because I am a beginner in metabolomics. In case, this procedure is right, that means that each feature will be processed under this step by step one to obtain a p-value because all features are expressed differently in the distribution and variance way between these groups (case and control).
I hope that you may spend a little time correcting my idea and give me some suggestions in this promising field.
Thank you so much.
Pham Quynh Khoa.
A PCR was run using 1/5 dATP, 1/5 dTTP, 1/5 dGTP, 1/5 dCTP and 1/5 dITP (where dITP is inosine and should pair with any of the other with some biases). Is there a quick way to check whether the inosines are being incorporated without cloning and sequencing the products to look for error rates?
Principle depends on the addition of acid or base at the beginning of the water extract of tobacco
For some applications in AUC, we would like to correctly determine the partial specific volume of our biomolecules.
We currently use Sednterp as a principal approach which diplays a theoretical value.
Is someone has a nice protocole to share to calculate partial specific volume on a densitometer? We use a DMA 5000 Tm as a densitometer.
I was determined protein carbonyl content by derivatization with DNPH. I take absorbance at 370 nm. Now I want to calculate in nmol/mg protein using an absorption coefficient at 370 nm of 22,000 M-1 cm-1. But I do not understand this calculation. So, I need cordial help. Thanks in advance.
I am going to fabricate a free flow electrophoresis (FFE) chamber. The chamber will contains the sample plus buffers (electrolytes). In capillary electrophoresis, IEF or gel-based electrophoresis the electrodes are in touch with buffers, because in those systems usually there is no external forces/pumps for buffer movement and electrochemical current needs to enforce the ions separation in designed direction that may not be straight.
However, in (FFE) system the buffer flow will be carried out by external pump and the electrical repulsion will be applied in perpendicular of sample flow which both are in a straight direction. So, is it also necessary in free-flow electrophoresis system that the electrodes be in touch with liquids (buffers) or not? in the other words, if the anode/cathode electrodes doesn't be in touch with buffer, the electric field/potential is still there and it could pushes the ionic particles in a straight direction towards opposite poles, is it true?
I am looking for a good reference book for students explaining techniques used in analytical biochemistry.
We used to use a book by David Holmes & Hazel Peck.
Any suggestion appreciated.
I would like to know the various techniques/methods which are used to characterize the crystals of a known substance (xylitol) and to check the purity as well as to quantify the impurities present in a crystalline compound such as xylitol (a sugar alcohol).
Eagerly waiting for the replies..
I've used the procedure proposed by Marrapu et al. (2015) -->
Is there any researchers who faced the same problem for short chain PCs using this protocol? What could be the problem?
We are trying to collect a sample before and after exercise. Plasma Dihydroxyphenylalanine (DOPA) is a precursor of urine free dopamine. If one collects the urine sample then what is the time frame to match your urine dopamine concentration to plasma?
We have used chloroform for cleaning and for extraction of used N hexane. We have also tried absolute ethanol. But quality and purity of oil is not good.
Kindly help me in this regard
I've used hexane to extract the oil from fish silage. Currently, I'm having fish oil mixed with hexane. There is a strong smell of hexane in the oil.
What is the best method to recover the oil from the solvent mixture (n-hexane)?
Is it possible to recover the oil by using anhydrous sodium sulphate?
I'm glad if anyone could help me in this?
Good day to everyone.
Can somebody provide me please, to get the answer to this question?
What is the reason for the number of wells in Microtiter plate that uses in Elisa technique is 96 wells or its multiples in some cases? Nor more neither less of that.
Thank you! I appreciate the help.
As we all know that lower Kd (micro-molar/nano-molar range) are considered as good interaction. But how one can appreciate the relative concept of this strength when I am measuring a single pair of protein-protein interaction. Please provide your valuable remarks on this. Thank you in advance.
I am working with an enzyme that makes 2-5 A chains from ATP. I am assuming the chain are 4 adenine residues long(no way to find out for sure; can't do mass spec). I am able to figure out how much ppi (pyrophosphate) is made when I mix enzyme with ATP... So how do I find out how much 4 residue long 2-5 A chains are being made? Using avocado's number?
Hi, I am trying to measure superoxide levels using EPR with DMPO as a spin trap. I had tried in isolated mitochondria and I got nice spectra (only in presence of inhibitors). Now I like to use total tissue, kidney and heart. If any of you have tried this can you provide conditions such as buffers and how you homogenize the tissue.
Also, anyone has experience in injecting spin trap into animals and measuring EPR later ? Ideally I want to perform in vivo measurements, or something close to that.
Protein mixed with iron sulfate in 100mM MES buffer, pH 6.5.
I want to detect various neurotransmitters in various biological samples. Please suggest a simple, accurate and economic method for determination of these neurotransmitter. like UV method.
It is very important in my research work. Please.
1) I am performing MTT assays and was wondering how much the cell size can influence the mount of metabolized MTT?
I thought that the amount of mitochondrial membrane, (so also the amount of active enzymes that metabolise MTT) correlates with cell size and therefore lets say 100 normal sized cells could metabolise approx. the same amount of MTT than 160 smaller cells, right?!
2) does anybody know if and how the metabolism of nutrients (Glucose, aa ) is connected to the activity of the MTT metabolizing enzymes ?
As maybe the presence of nutrients at the time point of the addition of MTT !!! can influence the capacity of metabolizing the chemical ..... (I mean : 100 cells consumed a lot glucose --> remaining glucose in medium is less and metabolism goes down...so also the metabolism of MTT, ..... smaller signal,.... but treated cells, where only 50 are present consumed less Glu, therefore still more in the medium when adding MTT and therefore 50 cells can metabolize more than 50% of the MTT than 100 untreted cells)
Thanks a lot for reading and trying to understand and helping :) !
i want to use QIAamp DNA Extraction kit for frozen stool samples.I have a Question,by this kit can i get good yield ?I,m looking Microbiota species .Can i find this species in this kit.
I have a 1D gel that I want to use for in-gel digestion. However this gel has been dried and placed between acetate sheets. In order to re-swell the gel and remove the acetate sheets I incubated the gel in 10% glycerol overnight. I then cut out my gel spots of interest and used the Shevchenko in-gel digestion method, dried down the peptides, re-suspended them and did a C18 spin column clean up. I wish to analyse these peptides by ESI using a QE mass spectrometer. Will the glycerol interfere with this, or will the in-gel digestion method followed by C18 clean up be sufficient to remove glycerol from my samples.
Thanks for the advice!
It is well known that Strawberry leaves contain high amounts of diverse phenolic compounds (Kårlund et al. 2014), and also polyphenolic compounds are known to interact with proteins and can inhibit enzymatic activity (Dawra et al. 1988; Suryanarayana et al. 2004).
How can I avoid high levels of polyphenols in strawberry leaf extracts? These high levels of polyphenols interfere with enzymatic activity.
What kind of techniques or protocols do you use to purify raw strawberry leaf extracts and eliminate polyphenols?
Thank you very much!
Dawra, R. K., Makkar, H. P., & Singh, B. (1988). Protein-binding capacity of microquantities of tannins. Analytical Biochemistry, 170(1), 50–53.
Kårlund, A., Salminen, J. P., Koskinen, P., Ahern, J. R., Karonen, M., Tiilikkala, K., & Karjalainen, R. O. (2014). Polyphenols in strawberry (Fragaria× ananassa) leaves induced by plant activators. Journal of agricultural and food chemistry, 62(20), 4592-4600.
Suryanarayana, P., Kumar, P. A., Saraswat, M., Petrash, J. M., & Reddy, G. B. (2004). Inhibition of aldose reductase by tannoid principles of Emblica officinalis: Implications for the prevention of sugar cataract. Molecular Vision, 10, 148–154.
HbA1c estimation using boronate affinity method requires a blue dye conjugated Boronic acid. I would like to procure one or two commercially available molecules. Please suggest me one or more.
As you know, BSA is commercially available as 96% and 98% pure. It contains also fatty acids. Therefore, i'm wondering how to get BSA purified.
I am looking forward to design a drug against Mycobacterium tuberculosis, so what are aspects (please point out some examples) should I look for ? We can target some enzymes which are not involved in human cells, but what type of cystosolic enzyme should be a good candidate please specify.
I extracted RNA today via the trizol method. My 260/280 ratio is 1.95 while 260/230 is 1.44. I washed the pellet using isopropanol and ethanol. The cells grown and RNA extracted from were human cells.
I have somewhat shaky hands especially when anyone watches me extract RNA. However, I did not notice any debris enter my pipette tip during aspiration of the supernatant.
Would it be possible to continue and synthesize cDNA from this RNA for qPCR? Would the qPCR be accurate?
Attached: My Nano Drop
We need the reference electrode to carry out the electrochemical corrosion tests at max. 10 bar (H2S or/and CO2) and 20-150 °C.
I prepared a solution of Pesin in 0,1M HCl and I stored it at -20ºC. Due to the concentration of pepsin that I need in this solution, I need to prepare a lot of solution in comparison to the amount that I need to use each time. As far as I know, HCl is activating Pepsin, do I need to prepare the solution every time I need to use it or by aliquoting it and storing it at -20ºC it is ok? Is pepsin loosing its activity when stored in HCl?
We wish to desiccate protein coated polystyrene microparticles with some other proteins inside a microchannel. We tried doing so using normal air drying (non-sterile air, from a blower). But when reconstituting the efficiency was quite less.
What is the preferred method to desiccate such reagents such that they can easily be reconstituted using PBS etc?
Any assistance will be appreciated.
I am writing a script for Processing fluorescence spectra. In Order to make it useful for a large audience, I am collecting Sample Data of Export Files from fluorescence spectrometers of different manufacturers (Perkin Elmer, Varian, Thermo Fisher, etc.). Anyone who would like to contribute may record a fluorescence Emission spectrum (excitation 280 nm) plus an excitation spectrum (Emission 350 nm) of water with an available spectrometer and post the Output Text File Here with all necessary information on the Device used for recording (model, manufacturer,etc). If multiple Output File Formats are selectable, pleased provide a Sample of each.
Thank you all for your help! :)