hardp3 at mayo.edu
Sun Mar 12 18:43:08 EST 2000
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.
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.
More information about the Methods