Hi everyone, I would like to find out peoples' opinions on the need to do validation of protein expression in a proteomics analysis (by western blotting for example). I understand that this was necessary in the early days when 2D image analysis software was not as reliable as they are today. I also understand the need to do validation if you wish to confirm protein expression over a much larger sample size. However, if you only have, say, 5 biological replicates, and you have identified proteins that change in abundance using 2D gels, is it really necessary to do western blots for validation? If so, why? Thanks.
I also agree with Claretta, protein validation is also necessary. If you do not have an antibody, you may consider excising your protein spot, trypsinization and mass spectrometry to unambiguously identify your protein of interest.
Claretta is absolutely correct. In fact, we have shown that both underlying assumptions in 2D gel electrophoresis are wrong: many spots contain more than one protein and most proteins give rise to more than one spot (we have also shown a few unique benefits of 2DE, see http://www.ncbi.nlm.nih.gov/pubmed/23033477). This makes any quantification that is based only on staining intensity in 2DE and on the performance of excellent image analysis software a bit questionable.
Validation using antibodies (possibly on 2DE Western Blots) and/or quantitative mass spectrometry is really important for being able to draw conclusions from your data.
I've also seen variable staining behavior using either Coomassie or silver. Some proteins stain well with silver but not Coomassie and vice versa. Although my point may be moot if you are doing fluorescent CyDye DIGE and using three different stains (link to GE's 2D-DIGE products follows...) http://www.gelifesciences.com/webapp/wcs/stores/servlet/CategoryDisplay?categoryId=1103746&catalogId=10101&productId=&top=Y&storeId=11787&langId=-1
But fluorescent labeling does not help if the second...tenth protein in the same spot is also labeled. WB helps if the antibody is ok. Coomassie is very different with different proteins - from strong to nothing, linear range of silver is not so good it runs easily into saturation. MS is a problem because it will depend on the extraction of peptides from the gel which may be different for each peptide and you need a 13C/15N labelled proteotypic peptide to quantify. One very nasty but sometimes successful way is cutting the spots to quantify between the samples, digest in spot and the label with iTRAQ and mix the samples. Sounds terrible but may work for 2D gels better than for 1D gels. I can give more advice on this if you consider this way.
I would recommend a western blot only because it guarantees that it is the same protein that you think it is . (use of antibody) . If not western blotting, you could also do a Mass spec analysis to ensure that it is the same protein that is being expressed .
I think you can also validate the expression of your protein by mass spectrometry. You can cut the band of interest (or dot) where you think your protein is, and you can perform an in gel digestion. The resulted peptides can be analysed by LC-MS/MS.
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