I' ve never done a gel shift before,
it's about my first gelshift: I purified some GST proteins and designed the oligo which contains a binding motif for the GSTs, and I make it for psitive control. meanwhile I use nonsepecific oligo for negative control.
the result is , positive control worked well, but my trouble is there is band shifted in nagative control either, although the shifted band for positive control is stronger thanthe band shifted for negative control, but how can I just kick the nonspecific band out or just optimize the situation, cause I need to do screening by using these GSTs in future.
I think maybe I can change the amount of poly dI.dC
but what else I can do? I dont't know how to adjust the salt condition for reaction.or PH or sth.
please give me your precious advices, and thank you very much in advance.
reation buffer:12% glycerol, 24mM HEPES, 8mM Tris-HCL, 2mM EDTA, 1mM DTT.
for each sample add 50ng poly dI.dC, 1ug BSA.
Edited by dllinpat, 09 January 2010 - 01:25 AM.