EMSA Problems

[philip hardwidge]

What acryl:bisacryl ratio are you using?  You might try 75:1 and see if that helps any.
I've had good success with this using some large proteins from nuclear extract.

-Phil Hardwidge
Mayo Foundation

Anton Tutter (atutter at aim.salk.edu) wrote:

: > A previous post-doc in the lab developed an EMSA using a 50 bp probe.
: > I've been able to repeat this experiment.  Now I'm trying the same thing
: > but with a larger probe: 125 bp.  The protein complex that is being
: > tested is ~270 kDa total.
: > 
: > I'm having terrible problems with the Probe not entering the gel(4%
: > acrylamide, Tris K0Ac, EDTA buffer).  The signal from the well is the
: > strongest(even more than the free probe, which can be seen).
: > 
: > There's no shift being seen, and the probe alone(no added protein) is
: > getting stuck in the wells too.  The probe is made from a PCR reaction,
: > and I've tried both Phenol extractions and Qiagen Spin columns to purify
: > the products, nothing works.
: >