EMSA Problems

[Anton Tutter]

> 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.
> 
> Any ideas on what is happening and/or what else to try?
> 
> The only step I haven't altered is the T4-PNK labeling followed by G25
> Sepharose spin column purification, but this step doesn't affect the
> smaller probe.
> 

the probe purity is probably not the issue.  it sounds like you've got
aggregates forming which will be too large to enter the gel.  try adding a
small amount of detergent (0.01 - 0.1% NP-40) to the gel mix, the reactions,
or both.  detergent usually prevents aggregation but your complex may or may
not withstand EMSA in the presence of detergent. actual mileage may vary.

--anton

tutter at salk.edu