emsa with ebox probe - multiple bands shifted - (May/08/2008 )

Hi guys,

Is anyone else doing EMSA with an ebox probe?? I've been doing it for a while now. Each time I get multiple bands shifted. I thought that these may have been non-specific bands but now I'm thinking that it is the formation of multiple complexes.

Anyone any thoughts??

Sara

my probe is 24 bases long. there are other potential binding sites within the probe..

I have seen in a few papers that ebox probes generate multiple bands in emsa.

I haven't tried a supershift experiment yet. I would hope to use a Snail Antibody.

thanks,

sara

I was using 40 base probe that gave me multi bands (6). After I used 24 bases probe and centralize the ebox within the probe (to avoid other motifs), I get only one band. You may shift the position of ur ebox motif within probe to avoid other potential motifs and u can use as short as 18 bases probe.

I think that supershift will determine if u have non-specific or multiple complexes. if only one supershifts, the others are non-specific .

Regads,
MolBiol

Thanks for that. My eboc site is centralised in the probe. I've tried a supershit assay and while my bands don't supershift, I do seem to be getting antibody interference, the intensity of my bands are greatly reduced when I add antibody.

I have been using whole cell extracts, at my bosses request but I am now going to try to use nuclear extracts to see if it reduces the number of bands.

Thanks for your suggestions,

sara

I was using 40 base probe that gave me multi bands (6). After I used 24 bases probe and centralize the ebox within the probe (to avoid other motifs), I get only one band. You may shift the position of ur ebox motif within probe to avoid other potential motifs and u can use as short as 18 bases probe.

I think that supershift will determine if u have non-specific or multiple complexes. if only one supershifts, the others are non-specific .

Regads,
MolBiol