Dear Yang, You might try acrylamide instead of agarose, since the caging effect of the tighter matrix might keep the complex together better. I'm not sure why MgATP? If your protein only binds in the presence of ATP you might need a non-hydrolyzable analog? Your concentrations are also vastly higher than those usually used for gel shift assays. You might think that would gaurentee binding, but many DNA binding proteins need quite a bit of salt to be soluble at high concentrations, so your protein might all be stuck in the well. Actually, you can coomassie stain an agarose gel with that much protein on it - that should tell you exactly where the protein went. Good luck! Phoebe
---- Original message ---- >Date: Wed, 1 Sep 2010 14:55:07 -0700 >From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of yang li ><robertcatru...@gmail.com>) >Subject: [ccp4bb] How to optimize protein-DNA complex conditions? >To: CCP4BB@JISCMAIL.AC.UK > > Dear All, > Recently I am working on a protein-DNA > complex, and from running agarose gel, there is a > weak delayed band after the band of pure DNA which > indicates some DNA has bind to the protein though > the binding efficiency is low. Then I tried to > optimize the condition to increase the binding, but > it > did not work since the intensity of the delayed band > didnot grow. The condition I used is list below: > 1. the concentration of protein is about 1.5mg/ml, > buffer in Tris-Cl, PH 8 and NaCl. > 2. the running buffer for agarose gel is 0.5x TBE, > PH 8.3. > 3. different ratio of protein: DNA has tried, from > 2:1 to 1:2. > 4. different concentration of NaCl, MgCl2 and ATP in > reaction system have been added, but no significant > change. > I wonder if there is any way to increase the binding > efficiency? Is it possible to set up crystal plates > in this situation, with protein and complex > together? > Any suggestion would be appreciated! > Best > Yang ===================================== Phoebe A. Rice Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp
