Gel Shift assays - (Sep/28/2005 )
I am just about to start doing gel shift assays. I am starting off using purified his-tagged protein (25KDa) and and a 200bp promoter region (bacterial promoter). Does anyone recommend any particular kit to use? I know that Pierce and Promega both do one - any feedback on these kits?
Also, for the non-dentauring acrylamide gel bit. How far should I run my gels? Is a minigel system (Biorad) OK or do I need a full blown 10X 10 gel?
any helpful hints would be great.
works for me every time
if the supershift is stubborn the incubation time has to be longer, but otherwise it's all there
i have looked at 2 different eukaryotic transcription factors in 8 promoter regions in several hundred samples and had pretty good luck, it works very well about 90-95% of the time and it's cheap
So you use agarose gels. Do you think this will be OK for bacterial transcription factors?
i don't know, but I don't see why not.
I look at NF-kB and AP-1 in human cells and it works like a breeze
i am assuming the same principles will apply with bacterial TF's
I started with agarose because it was more familiar to me (I have run many many southerns, very few westerns )
it worked well, so I stayed with it.