I expressed protein in P.pastoris and performed zymogram (to check enzyme activity in gel) and did western blotting using Nitrocellulose membrane, in both band observed was in same position (around 45-50 kDa but my expected size of protein is 34kDa).
Later I did western blotting using PVDF membrane; protein band in zymogram was same as earlier but after western blot I got expected protein band size on PVDF membrane.
Now I am confused why activity band in zymogram and protein band after blotting in PVDF are of different size?
Does PVDF somehow helps in deglycosylation ? I repeated it 3 times with PVDF and got same result.
Hope anyone could help me. Thanks !!
In your case it is more likely that your protein is not stable, there is a loop region in the structure around 34kD where some of your expressed protein molecules are degraded inside the expression host by cellular proteases, your SDS-PAGE will show a ban of full-length protein and a band of degraded protein. Different Glycosylations of your protein do alter the protein size, but usually with a variation around 5 kD.
Thanks Liu for replying but variation in my protein band size more than 5kD. Could you please suggest how to resolve this issue ??
In single SDS PAGE gel i run my protein in two lanes usually, I cut out and use one lane for activity staining and remaining for western blotting. From same gel I get different size bands (45-50 kDa) from activity staining (zymogram) and in western blotting (my expected size 35 kDa) that's why I am very much confused !! I hope I am able to explain the issue.
If you want solve this problem, you need to test if that is the case. What you need to do is to do expression trials at different temperatures. Usually you can get more full-length at a lower temperature.
Most protein degradation occur at the C-terminus, if you fuse a affinity tag at the C-terminus of your protein, you might be able to purify the full length version even with some degradation. However, this kind trouble-shooting is always in a style of "try and error", you can only work it out after trying with different approaches.
Dear Liu, thanks a lot for your suggestion. I will try doing at diff. temp.
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