Hi, Kevin, Wash your crystal very very well to get rid of unbound DNA. Then run your crystal with SDS PAGE gel, with protein and DNA as markers. After staining the gel with silver stain, the DNA will show with different color and migrate about where the dye locates. So don't run too long time.
Regards, Nian On Tue, Jun 23, 2009 at 3:38 PM, Kevin Jude<[email protected]> wrote: > Note that, despite the claim otherwise in Kettenberger and Cramer, SYBR Gold > does stain at least some proteins, so be sure to run the appropriate > controls. > > kmj > > On Tue, Jun 23, 2009 at 11:52 AM, Allyn Schoeffler <[email protected]> > wrote: >> >> Dear Nick, >> >> If you have access to a fluorescent microscope, you can try staining >> crystals with PicoGreen and seeing if they fluoresce. (see ref: >> Kettenberger H & Cramer P, 2006, Acta Cryst D v62 pp146-150: >> Kettenberger H, Cramer P.Fluorescence detection of nucleic acids and >> proteins in multi-component crystals.) >> >> I've had good luck harvesting & dissolving crystals and then running a gel >> (stain with SYBR-Gold to increase sensitivity). If you can't get enough >> material to detect a band, you can radioactively kinase the dissolved >> crystals and then run a gel. >> >> Best Regards, >> Allyn >> >> >> >> > Dear all, >> > >> > I am trying to solve the structure of a transcription factor in complex >> > with its DNA. I got crystals of the complex under different conditions >> > than the protein alone and they also look different. Unfortunately, they >> > only diffract to 6Å so far. Before I continue to optimize the crystals I >> > would like to confirm that the crystals really contain the bound DNA. >> > Thus I tried to crystallize the protein alone under the same conditions >> > as the complex which did not give crystals. I also tried to stain the >> > crystals with methylene blue, but that did not work (but staining with >> > IZIT did not work either). Additionally I dissolved a crystal and >> > measured the absorption. The ratio between A260 and A280 was 1.3. So >> > there seems to be DNA, but less than there should be. >> > >> > Does any of you know a good way to quickly but reliably confirm the >> > presence of DNA in my crystals? >> > >> > Thanks a lot in advance. >> > >> > Nick >> > >> >> >> -- >> Allyn J. Schoeffler >> Berger Lab >> Dept. of Molecular and Cell Biology >> UC Berkeley >> phone: (510) 643-9491 > > >
