Currently, I am working with two recombinant versions of the protein A (SpA)- one with cysteine and the other without cysteine. On coomassie staining in both cases, the band intensities are always the same with respect to the amount of protein that is loaded onto the SDS gel. Whereas on silver staining, only the variant with a cysteine attached to it is able to develop the silver stain, and that too is not of very high intensity. But the one without cysteine is not at all visible after silver staining. I have tried the experiment 3 times taking BSA as a positive control where BSA gets silver-stained properly every time but the variant without cysteine gets never stained. What could be the conclusion for the whole result? And to what amino acids do the silver ions actually bind?
Hello Piyush Singal
Silver staining technique involves the deposition of metallic silver onto the surface of a gel at the locations of protein bands. Silver ions (from silver nitrate in the staining reagent) interact and bind with certain protein functional groups. The strongest interactions occur with carboxylic acid groups (Asp and Glu) and sulfhydryls (Cys).
This technique is based on the simple principle that selective reduction of silver into metallic silver occurs at the initiation site in the close proximity of protein molecules. However, lack of staining indicates that proper protein density and charge are prerequisites for silver-protein complex formation. Silver ions are bound to negatively charged groups of proteins.
Please refer to the references below for more information.
Article The mechanism of silver staining of proteins separated by SD...
Article Mechanisms of protein silver staining in polyacrylamide gels...
Best.
"lack of staining indicates that proper protein density and charge are prerequisites for silver-protein complex formation"
so this mean that your cys-SpA is better expressed than the wt-SpA or that it has a better charge (du to the extra cys)? or both ?
Hi Didier Poncet
I believe that the SpA with cysteine has a better charge as compared to the one without cysteine.
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