EMSA - probe design and competition - (Jul/12/2010 )

Hi,

The lab that I am working in is really new to EMSA and functional genomics as a whole. Part of my thesis project is to design an EMSA study for a particular SNP in the TAGAP gene. I've got protocols for EMSA both radiolabelled and non-radiolabelled. I'm having trouble finding advice on how to design my probes.

How long should my probe be? Is this dependent on how big the presumed transcription factor is?

Also - how do I design competitor probes? How many competitor probes do I need? Is there a protocol somewhere that will tell me this?

I appreciate any and all advice.

Liz

You can look into the literature to find out how people do such experiment. I actually did similary EMSA to study a snp. Regarding probe size, something around 20 nt is good enough. competitor is an oligo with unrelated sequence. One is good enough, of course the more the better.

I used to do competition EMSA with both "specific" competitor (unlabeled probe that is identical to your labelled probe) and "nonspecific" competitor (as pcrman described). In addition, you can use a series of unlabeled competitor oligos with different mutations in the putative binding site, if you're interested in that level of detail.

epibio on Jul 13 2010, 11:22 AM said:

So does the sequences for the nonspecific competitor need to include anything specific (like a TATA sequence or a certain percentage of GC)- or is it just random sequence?

lizbit on Jul 13 2010, 12:31 PM said:

It doesn't have to, it can be random. In my case, I used sequences that contained binding sites for other, unrelated transcription factors, simply because I had them already in the lab.

Thank you both so much! You've been a huge help!

Liz

I am doing EMSA first time.Can you please tell me how to anneal the oligos? I am using 50 mM tris,10 mM NaCl & 1mM EDTA and then heat for 5 min at 95C & cool slowly on heating block.But it doesnt work I cant sea annealed probes.Can you plz tell me the procedure for annealing?

Sata