Mobility shift assay

Giovanni Maga maga at vetbio.unizh.ch
Wed Dec 20 09:06:30 EST 1995


In article <v0151010aacfc914ee7b3@[128.118.22.149]>, rxr21 at PSU.EDU (Ramesh
Raina) wrote:

> 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

I am not sure you can detect a difference in shift of just 23 kD, using a
DNA of 250 bp. But anyway, since the DNA is quite large, are you sure that
you have a single binding event for each DNA molecule? I mean, if there
are three 47 kD or two 70 kD proteins bound per each DNA mol., you'll end
up with very similar shifts. It can be also that the protein needs to be
in a multimeric form to bind the DNA, so the 47 kD forms a trimer in
solution, whereas the 70 kD forms a dimer and in this form it binds the
DNA. You can check this by quantitating the amount of DNA shifted at
different protein concentrations and calculate the molar:molar ratio of
the DNA:protein complexes. Maybe you'll find something interesting.
Hope it helps. Giovanni.



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