EMSA Problems

P.J. Shaw pjs14 at le.ac.uk
Mon Mar 13 07:32:15 EST 2000



On 12 Mar 2000, philip hardwidge wrote:

> 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).

Try lowering the acrylamide concentration, increasing the acrylamide:bis
ratio or both. What buffer do you use? I found tris-glycine to be the best
for resolution, doesn't need to be re-circulated.

Phil Shaw.
pjs14 at le.ac.uk
 
> : > 
> : > 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.
> : > 
> 
> 





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