Hi, everyone.
I'm trying to purify an enzyme present in very low quantities. It's
almost pure now and the activity is still there, but I don't know which one
of the proteins I see in the silver-stained gel is the one I'm after. My
next step is to resolve the proteins by PAGE (denaturing and
non-denaturing), transfer them to nitrocellulose orPVDF, and ...(here is my
question) detect the protein of interest with the substrate?. I can use a
substrate that has been labeled, and do the binding at low temperature to
prevent the enzymatic reaction from happening, but of course, this requires
the active site to be in the right conformation. Has anyone done this or
something similar before, or know of any reference that I could use as a
guide? I know one can detect kinase activity by incubation of the nylon
membrane-bound enzyme with the right substrate, and detecting incorporated
radiolabel by autoradiography. Any other examples?
Thanks for comments.
Miguel Zamora, Ph.D.
University of Oklahoma
Health Science Center
800 Research Parkway
Oklahoma City, Oklahoma 73104
