Dear Researcher,
I am trying to imminoprecipitate (IP) Sumoylated protein. i am succesfully able to get it. now i have to carry out desumoylation of that immunoprecipiteted proteins in vitro. I tried to IP protein and treated with catalytic domain of SENP1 enzyme for 2hr at 37oC but it is not working. Do you suggest what mistake may be i am doing? I am adding 20 ul of buffer mixture (25mM Tris, 20mM Nacl, 5mM DTT) containing 300nM senp1catalytic domain?.
If the protein is still bound to antibodies used for immunoprecipitation, they may be blocking the access of the enzyme to the cleavage site.
Dear Adam B Shapiro. thank you very much for suggestion but what could be the solution for it?
@ Sanjay pH of buffer is 7.5 it seems optimal as per literature.
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