how much should the shift move above in case of supershift - (Mar/13/2007 )
i want to know how much above the shift should a supershift be. i have purified a protein and i have done a gel shift assay which i got. i have two types of antibodies, one against the tag of the fusion protein and another which i have got raised after peptide synthesis. should these two supershifts come at the same position or which one should be above if at all. and how to know that. i guess it should depend on the size of the antibody, so how to know the size of these antibodies especially in the latter case.presently i'm getting a very small supershift.
I apparently have the same problem ! I can see the gel shift, and when I add the antibody, I can only see a very very small shift (others can also see it, it's not only my eyes, wanting absolutely to see something ! :-)
I was adviced to add a secondary antibody, to increase the weight of the antibodies complex. I'm trying that today so we'll see !
I was also thinking that I should change the acrylamide % of my gel (I'm currently doing 5% gels for a 34 bp probe and a 105kDa protein, but it's certainly a complex of 2 proteins so probably much more). But I'm not sure if I should increase or decrease it and how much...
If anyone has clues !!
Thank you !