Gel shift with high isoelectric point protein - (May/15/2007 )
I have been trying to do non radioactive gel shifts using fluorescein labeled primers. The problem I have is that my protein has a high isoelectric point (8) or 10 if I use a SUMO tag protein, therefore the normal running buffers are not working because they are usually between 7 and 8.3 in pH. The only way my protein goes into the gel is with a Glycine buffer that has a pH of 10.6. I tried this buffer in a gel shift and my protein-DNA complex does not seem to be stable at this pH. I was wondering if anybody had done gel shifts with a high isoelectric point protein or if anybody had any ideas on how to keep my protein DNA complex stable at this pH. Any help is greatly appreciated.
I do BS as well but not in extrem conditions, lol,
what if you try to play around with salt concentrations to keep the interactions?