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Postby dhanyar1979 » Fri Apr 20, 2007 10:04 am

Hi... i am trying to isolate and purify a particular protein in fish muscle which is found in very small amounts... it happens to be an enzyme and wheni do the sds i am not sure if at all i get a band corresponding to protein of my interest ... and as i procced with my purification further.. i do not get any bands... thougt i get very ggood activity... is there any method in wchich we can see bands of proteins of very low concentration..

Thanks in advance for reply. My email id is [email protected]. you can also contact me in this id
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Postby blcr11 » Fri Apr 20, 2007 11:34 am

A western blot would probably be the most sensitive, but you most likely don’t have suitable antibody or anti-antibody conjugate. You could try silver staining, but that won’t be specific for your protein. You might try zymography if your enzyme has a substrate that can be put into the gel. I’ve not done zymography before, so I can’t say how difficult or easy it is to get it to work, but it ought to be specific for the target enzyme. If I had to do it, I would try and clone the gene and express it in either E coli or insect cells.

You're sure your assay is all right--no possibility of contamination of reagents, etc. It doesn't happen all that often, but there are known instances of proteins that just don't stain well with the usual coomassie stain.
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