EMSA Problems

jqpublic at home.net jqpublic at home.net
Sat Mar 11 15:12:28 EST 2000


Hi,

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.

TIA,

c-d






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