Check with your sample buffer. Boil the sample+sample buffer properly. By the way whats the molecular weight? Any idea?
Compare the protein's position in the gel with molecular weight markers to see if it is running in the expected place. If it is running at too high molecular weight it could be because it was not properly reduced and denatured. Make sure you are using a denaturing (SDS) gel not a native gel. Make sure your sample buffer contains the correct concentrations of SDS and reductant. Be sure to heat the samples before running them.
Hi Radha, just to add to the answers above, if your SDS sample buffer has less than 1% SDS, then it is possible that proteins run abnormally on SDS-PAGE.
I am sonicating cells in glycine buffer and even heating the sample buffer at 95 C for four minutes but still the enzyme molecules are not moving more than 2-3 mm below the wells in the stacking gel, other polypeptide chains are moving to the end of the gel. I have done activity staining as well when gel is immersed in PNPP containing buffer it is showing yellow bands below the well.
If you are seeing catalytic activity in the bands, then the protein was not denatured. You may have omitted SDS or reducing agent.
change the percentage of gel. the protein seems to be of a higher kDa. 10% would be better. or over run it and confirm.
Dear Mr Adam, Masood, Stefansson and Raghu Ram, Thanks for your reply.
When i am heating my samples and using higher amount of SDS protein bands are appearing more clear and even moving in the resolving gel. But how then activity staining will be performed?
Remove sds using triton x100 and perform the activity as you do always.
Rather than using in-gel activity, perform the enzyme reactions in solution.
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