Gel Shift assays - (Sep/28/2005 )

Hello all,

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.

cheers

http://www.protocol-online.org/prot/Detailed/3500.html


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

Thansk Amiee,

So you use agarose gels. Do you think this will be OK for bacterial transcription factors?

cheers again,

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.