I expressed my GOI in P.pastoris and deglycosylated the expressed protein using a kit and got expected size band. Now I am planning to do a western blot with deglycosylated protein. Therefore, I wanted to ask whether I do western blot directly with deglycosylated protein without further purification? Do buffers, enzymes from deglycosylation kit might interfere with western blot??
Since you were able to run the deglycosylated material on SDS-PAGE without a problem, Western blotting should be OK also.
Hi there,
I don't think there is any problem using deglycosylation mixture for Western blot. This mix contains nothing but proteins in a very usual buffer system.
The effectiveness of a particular Western blot also depends on a nature of respective antibody, i.e. what kind of antigen was used for an immunisation protocol. According to my experiences, deglycolsylation chemicals in the kit didn´t interfere with the Western blot protocol.
Mr. Ashraf, could you please elaborate on Dos and donts please.. Thanks
You can use do your western blotting with deglycosylated protein and is going to be perfect. Kits buffers and enzymes will not interfere with your western blotting analysis unless upon contamination or other things.
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