The simple answer to your question is 'no'. Even a single spot on a 2-D gel can contain several to many proteins. With respect to the ambiguities mentioned above, the more important or significant a protein identification becomes, the more important it is to carefully scrutinize the software search results. The more you want to believe a result, the harder you should work to make sure it's correct.

Thank you Steve. BLAST will produce so many more hits especially when the stringency of the search is wide. Such was my recent experience with (an) extracted protein (s) from Archachatina marginata. The available databases for the BLAST did not contain information on the snail, so a wide stringency BLAST search was made. The hits were so amazing!

In your band there are usually more proteins especially when the sample is complex . You can use a 2d or you can narrow the bioinformatics parameters. But in a band keep in mind that there may be more protein and therefore can be significant even more hits.

Even without BLAST and the multiplicity of answers it creates, an SDS-stained gel band almost invariably contains more than one protein. In our lab we routinely do nanoLC/MS/MS of gel bands, and we rarely see less than 20 proteins identified from a band; sometimes well in excess of 100, and this is with MASCOT database searching that will only really find identical sequences. If your interest in the band originates from e.g. a Western Blot of the same, this can indeed be problematic as the reactive protein is often not the most abundant in your gel band.

From my own experience I would advise caution in using wide stringency BLAST on this type of data; make sure you have very high quality MS/MS spectra on the proteins in your sample, manually check the quality of any de novo sequences that are obtained, and make sure that the result is at least plausible with regard to isolation procedure and molecular weight of the protein candidate. the way BLAST scoring works it is difficult to work with samples of even moderate complexity.

Now that BLAST, MASCOT, Scaffold(R) and other softwares are making protein structure determination so confounding, how were the earlier biochemists able to come up with so definite structures of the known proteins? Are we retrogressing in our quests as biochemists? It looks like the best we can get from bioinformatic tools are mostly guesses!

Not guesses, but we are often dealing with probabilities rather than "solid" results. MS/MS data are in many regards more ambiguous than a nice Edman sequencing ladder; they are, however, also potentially order-of-magnitude more powerful. The important thing is to be aware of the ambiguities.

The simple answer to your question is 'no'. Even a single spot on a 2-D gel can contain several to many proteins. With respect to the ambiguities mentioned above, the more important or significant a protein identification becomes, the more important it is to carefully scrutinize the software search results. The more you want to believe a result, the harder you should work to make sure it's correct.

Dear Musa

If you use SDS-PAGE only, it is possible that one band contain some different proteins with similar Mw. Also, in the case of 2-DE, the purity of your spots is stricly dependent to the complexity of your samples and conditions of 1st dimention of electrophoresis.

Thank you Masood. I have seen that analysis of protein using electrophoresis and other methods is a complex exercise. And I have always asked myself how the earlier biochemists have done it to have come up with such vivid structures of the known proteins when isolation of a single pure protein can be such a big challenge.

Dear Musa

Protein methods are the techniques used to study proteins. There are experimental methods for studying proteins (e.g., for detecting proteins, for isolating and purifying proteins, and for characterizing the structure and function of proteins, often requiring that the protein first be purified). Computational methods typically use computer programs to analyze proteins. However, many experimental methods (e.g., mass spectrometry) require computational analysis of the raw data.

I work with proteins since 2008 and published some paper on it. At this time, I work on 2DE-MS (one technique in proteomics). If you need some helps about protein related techniques (such as chromatography, SDS-PAGE, 2DE, protein expression and purification) or want to do study about proteins, please dont hesitate to contact me

Cheers

Thank you Masood! I look forward to working with you in the near future.