Mobility shift assay

Ramesh Raina rxr21 at PSU.EDU
Tue Dec 19 14:22:49 EST 1995


Dear Nitters,

I am having some trobule with mobility shift assays. I am using a 70 KD
protein and a 250 bp DNA fragment . I get a nice shift. When I use a
deletion derivative of this protein (47 KD, which retains DNA binding
domain), with the same peice of DNA I do get the shift but the shifted band
migrates similar to one with full length protein (70 KD protein). I would
expect the DNA-protein complex with 47 KD protein to migrate faster than
the complex with 70 KD because the DNA fragment used in both the cases is
same but protein sizes are significantly different. I am using a vertical
4% PAGE. I have tried both 40:1 and 80:1 acrylamide:bis ratio. Could
someone suggest me what is going on and how can these complexes be
resolved?

Thanks in advance.

Ramesh





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