western blotting - transferring a small MW protein

john.hines at yale.edu john.hines at yale.edu
Fri Mar 22 22:05:09 EST 2002


Hey everyone,

   This is the first time that I have ever tried to do a western for
something so small.  My target protein is 10 kDa.  I'm used to larger
targets (40 - 100 kDa).  I'm doing wet transfer in the standard
Tris/Glycine/SDS/methanol buffer from out of Harlow and Lane.  Pure
nitrocellulose with the .45 micron pore size.

    What I'm looking for are recommended transfer conditions.  How many
volts (or milliamps) for how long a time?  I don't want to blow my tiny
protein through my nitrocellulose, but at the same time I want to get as
much of it onto the membrane as possible.


    Does anyone out there with experience in western blotting for smaller
proteins have a place to start?


John

john.hines at yale.edu




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