Enzymatic assays to detect for the protein activty have shown activity of interest is present but running the gel (5:5) sample buffer /protein sample shows no bands on the gels with both coomassie blue and silver staining . This is even after carrying out fixing in 40% methanol and 10% acetic acid
This could mean that your protein concentration is very low but activity is high. Did you quantify protein (Brdford/BCA/Lowry) before running the gel?
You are getting enzyme assay positive because of high specific activity of protein. If you are not getting any protein band after silver stain that mean protein conc. is too low. To get zymogram, you should load appropriate amount of protein to gel. It will be better to re-extract the protein of interest from sample, may be with higher volume of culture and then concentrate the protein by ammonium sulfate/Amicon filters. Once you will see bands (native gel) then go for zymography with optimal pH and temperature conditions.
Best wishes..
Hi Lydia,
Is your protein acidic? In acidic buffer conditions, coomassie dye binds to basic and hydrophobic residues of proteins. But, a number of acidic proteins are poorly resolved on acrylamide techniques gels using Coomassie blue or silver nitrate staining.
Perhaps this paper I have attached is useful for you.
Best wishes.
Yes the protein is acidic..... Thank you all for your insightful comments!
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