The same goes for other DNA-binding dyes. I've seen a polymerase that binds EtBr. -Joel
On Tue, Jun 23, 2009 at 1:38 PM, Kevin Jude <kj...@berkeley.edu> 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 > <asch...@berkeley.edu>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 >> > > >
