To quantify a protein under experimental conditions, western blot is used as a major tool. The data is then represented as the level of the same protein and then correlated with its functional level. At the same time western blot has no or has a very poor (if the antibody targets any sequence in the active cite or phosphorylated site of the protein) correlation with the actual level of the functional protein. Therefore, the above technique is now going to replaced with mass spectrometry or tagged protein assays. In that case, does anybody believe that spectrophotometric assay of enzymes or any protein gives the accurate data about the functional protein level than western blot analysis?
Here you mention the specific case where the active protein is the phosphorylated form of the protein, which holds true for many proteins in signaling pathways. If you are comparing phosphorylation of your favorite protein between different conditions, it is ultimately possible to do so by western blot if you have reliable specific antibodies against the protein (targeting an epitope that is not affected by phosphorylation) and to the phosphorylated form of the protein. Depending on the quality of your antibodies you could compare the phosphoprotein/protein ratio between conditions (but not the absolute amount) by comparing each form to a housekeeping gene.
However, there are also the cases where the active form of the protein is a multimere or in interaction with other proteins, and a variety of other circumstances. It could also depend on specific combinations of modifications other than phosphorylation. In such cases, I don't think western blot would be an appropriate tool.
Ultimately, if your protein is an enzyme, I'd choose to do an enzyme assay. That'd be the most straightforward option if you're only interested in the active form. But in the case where the enzyme is modulated by phosphorylation, western blot/mass spec experiments would be desirable to complement the results. However, if you do want to compare the active/total protein ratio, made spec is the most robust option if the activity is regulated by PTMs, specially if the protein has several modification sites.
Western blots are only as good as the antibodies you are using. For new proteins MS based quantition should be superior.
Here you mention the specific case where the active protein is the phosphorylated form of the protein, which holds true for many proteins in signaling pathways. If you are comparing phosphorylation of your favorite protein between different conditions, it is ultimately possible to do so by western blot if you have reliable specific antibodies against the protein (targeting an epitope that is not affected by phosphorylation) and to the phosphorylated form of the protein. Depending on the quality of your antibodies you could compare the phosphoprotein/protein ratio between conditions (but not the absolute amount) by comparing each form to a housekeeping gene.
However, there are also the cases where the active form of the protein is a multimere or in interaction with other proteins, and a variety of other circumstances. It could also depend on specific combinations of modifications other than phosphorylation. In such cases, I don't think western blot would be an appropriate tool.
Ultimately, if your protein is an enzyme, I'd choose to do an enzyme assay. That'd be the most straightforward option if you're only interested in the active form. But in the case where the enzyme is modulated by phosphorylation, western blot/mass spec experiments would be desirable to complement the results. However, if you do want to compare the active/total protein ratio, made spec is the most robust option if the activity is regulated by PTMs, specially if the protein has several modification sites.
Yes, I agree with both of you comments. But if you will go through the literature then most of the instances are there where people correlated the protein level using western blots. Sentences such as "the transcripts data are well correlated with the expression of the protein level (by western blot)" will be found where the meaning is the transcript level is correlated with the level of its corresponding protein. I think it means the active protein irrespective of its functional level is correlated in those articles. So can we say it is a not completely true sentence in context dependant manner.
If your 1st antibody has very good specificity, and if you use fluorescent 2nd antibody, I think it should be good for protein quantification.
Add a known quantity of pure protein to your sample in, let's say, five different concentrations and run your blank sample next to it. This should account for all errors in your experiment and give the best concentration you can possibly detect.
Biswaranjan, I'm not quite sure I'm following you. Your distinction between "total protein" and "functional protein" is not very clear, especially your comment about the antibody "targeting the active site". An enzyme has a catalytic site whether it is active or not. Targeting that site will not solve the issue.
But first things first: most proteins do not have two distinct functional/non-functional forms. It is true for specific proteins that require PTMs or increased levels of calcium to function, but most proteins are "functional" in the presence of interacting partners and properly localized. In that case, distinguishing the "functional and non-functional forms" of the protein is pretty impossible.
Second, when authors use western blot or any other technique to quantify the expression level of a protein, I think they they're doing just that. Even if the protein needs to be activated, localized or to interact with other proteins to have any effect on cell behaviour, the first step is always to check whether there is any variance in the expression level of the protein, and western blot can answer that. The issue of the activation, localization, interactions, etc can be addressed subsequently.
If your concern is that authors are jumping to conclusions by directly associating increased expression level with increased action of the protein, keep in mind that many proteins do not need to be activated, just expressed, and that when it is known that the protein requires other factors to function, this is usually checked in separate experiments. Or at least it should.
Ya thats true. But for quantification of enzymatic proteins do the above explanations hold true? Because at the end of the day you need to correlate the enzyme function (if measuring by WB and many of the papers do not measure the activity at biochemical levels) with the experimental condition(s). And I think WB has no or a little connection between enzyme function rather the protein level. PTM, cofactors, co-partners etc. for proper functioning of an enzyme can not be measured by WB. In many papers the instance of the above fact is known.
It ultimately depends on the question you are trying to answer. Of course, the most straightforward way to detect a change of enzyme activity between conditions is an enzyme assay. But if you detect a change, then the next logical question is, why is there a change? Is there more of the enzyme (for many if not most enzymes, that will be the case). Or has the enzyme been activated by a PTM or a change in concentration of a metabolite or ion? Then, you'll need a western blot or other technique to check that. So you'll have to check the enzyme quantity alongside its activity anyway. Also remember that enzyme assays employ optimal conditions to detect enzyme activity (excess of substrate, etc). But in vivo, it could be that the increased enzyme activity is compensated by something else (enzymes catalyzing the opposite reaction, disadvantageous concentrations of substrate/product, etc) and all of that has to be checked. So an enzyme assay does not tell you everything you need to know. Other techniques, such as the western blot, have to be done alongside anyway. And of course, everything will be adapted depending on the exact question you want to answer.
There are three types of westerns (nice to see everyone using the correct, lower case form 'western' here) - 'the good, the bad and the ugly'. We should accept that 'standard' westerns, as performed in a typical lab, are barely quantitative. Some newer approaches might well be. And, as the correspondents above have said - we must not equate protein amount with protein activity.
But, and this is a big 'but' - mass spectrometry cannot even approach the sensitivity of immunological methods - even with careful SRM assays (10amol for a S/N of 10 would be a triumph). So, the trick will be to improve westerns, yet retain sensitivity. That will at least give you one number (amount of protein) to start the understanding.
And, however difficult it is - it is better to assume expression changes at the protein level than at the transcript level. So, at least, we'll be working in 'protein space'.
Enjoyed reading the above comments. I would rather equate the amount of protein seen in blot (say a cell lysate transfected with different amount of plasmid--titration experiment) with its activity (whatever it does), by measuring them in a parallel experiment using the same sample. By this way one can at least, relate/compare the relationship of a protein's quantity with its corresponding activity. Most of the antiviral proteins we work follow this rule, by titrating different amount of plasmids with the inhibitory activity of a particular virus.
I see a confusion of terms in this discussion.
The question was: comparison of MS and blotting to get "accurate data about the functional protein level". Why people talk about enzymatic activity? Further, I hope we are talking here only about RELATIVE quantitation.
MS quantifies proteins on the level of peptides produced by protease digestion. The functional status (is enzyme active or not) does not matter. Usually, several (preferably many) peptides per protein are quantified. Here, you may have partial protein degradation, different levels of processing, inconsistency in digestion, post-translational modifications (like different levels of met oxidation, deamination, etc) between samples so it is very often happens that for 3 peptides from the same protein you see ratio 1:2, for another one 1:3, and for 3rd 1:4. Then AVERAGE ratio is calculated. You see that the protein is up-regulated but how real the value is? However, it is a correct estimate if you quantify by MS.
When you use blotting, you detect the whole protein. Enzymatically active or not, it is again does not matter if you use SDS-PAGE. The blotting can be quantitative, at least semi-quantitatitve for relative analysis - you just make a calibration curve: 25%, 50%, 100%, 200%... depending how drastic is the difference between samples. After detection, you analyze the intensities of bands by one of the imaging programs. It is very often done and is accepted for papers, you just take care that the calibration is done correctly.
And it is very possible that if you analyze the SAME samples by both MS and blotting, your estimated up-regulation values will differ! But in both cases, the protein will be up-regulated.
And, both values will be correct, and accurate, taking into account the method used.
By the way, I don't advice to believe in correlation between mRNA and protein levels.
Especially in exact values of differential regulation. This is simply not true because of many reasons.
Hey, Natalia, I completely agree with your view. However, if one will consider the quantitated intensity of one enzyme in western blot, will it be correct when the activity of the protein is discussed. The activity of a protein is a major character to be considered for its functional level. In western blot, a part of the protein is detected by antibody which of course corresponds to the whole protein but having a very poor correlation with its functional level. Is not it. Nevertheless, in large number of papers, a protein (enzyme) level is also relatively quantified and discussed in western blot. Is it correct?
If it is activity of the enzyme what is under the question then it should be measured by the activity assay. And, when you characterize your enzyme, you actually should provide a so called specific activity value, amount of activity units per mg of the protein.
When you isolate the enzyme in the active form, different preparations (lots) can have different specific activities.
Whenever the protein, the enzyme or not, is assessed by western blot, it is protein amount that is detected.
Another thing if it is known that, for example, the active form of the enzyme is the phosphorylated form, and the inactive is dephospho form. In a way, you can argue that if you quantify the presence of the phosphorylation, you quantify the activity. If you clearly state it in the paper, it most probably will be accepted because then it is evident what you measure. But strictly speaking, it is not absolutely correct because activity depends also from other things (proper folding, stability in the buffer and other things) so the activity assay is again the only way to be sure.
It is a protein level that is quantified by western, not the enzyme activity.
When you read a publication remember that they are written and reviewed by humans. It might be that the author simply used words "protein" and "enzyme" as synonyms.
Yes, thats what I was pointing in my question what you have mentioned in the last part. May be thats a human error but many papers have that and it seems as if they want to say about the enzyme activity not only about the protein level measured by WB.
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