atutter at aim.salk.edu
Sun Mar 12 00:58:16 EST 2000
> 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.
tutter at salk.edu
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