salt condition of EMSA - I need to adjust salt condition to improve the specificity of my EMSA (Jan/08/2010 )
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.
See this Trouble shooting
download at www.roche-applied-science.com/pack-insert/1635352a.pdf
or get from my message
Aslo there are a lot of binding buffers, I can show some
buffer 1) Biswas S, Biswas I. Regulation of the glucosyltransferase (gtfBC) operon by CovR in Streptococcus mutans. J Bacteriol. 2006 Feb;188(3):988-98.
buffer 2) Senadheera MD, Guggenheim B, Spatafora GA, Huang YC, Choi J, Hung DC, Treglown JS, Goodman SD, Ellen RP, Cvitkovitch DG. A VicRK signal transduction system in Streptococcus mutans affects gtfBCD, gbpB, and ftf expression, biofilm formation, and genetic competence development. J Bacteriol. 2005 Jun;187(12):4064-76.