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Electrophoretic Mobility Shift Assay Forum EMSA Forum. Discuss the Electrophoretic Mobility Shift Assay protocols or technique and the assessment of DNA and RNA binding proteins here. Also post questions and troubleshooting about the shift assay.


specific protein-DNA complex staying up in wells in EMSA

specific protein-DNA complex staying up in wells in EMSA - Electrophoretic Mobility Shift Assay Forum

specific protein-DNA complex staying up in wells in EMSA - EMSA Forum. Discuss the Electrophoretic Mobility Shift Assay protocols or technique and the assessment of DNA and RNA binding proteins here. Also post questions and troubleshooting about the shift assay.


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Old 04-28-2011, 02:36 AM
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Unhappy specific protein-DNA complex staying up in wells in EMSA



Hi all,

This is my first post here. I'm having trouble with EMSA and I was hoping you could help me. I'm doing EMSA with 10ug of nuclear extract got using the Thermoscientific Pierce kit, and using 0.5uM of a 150-260bp PCR amplified and gel purified or column purified 5'6-FAM labeled product as my probe. The probes have been eluted in Tris-HCl, EDTA and Nacl, and HEPES or Tris based binding buffer used for the reactions. After 30min incubation at RT, I load half or more of the reaction volume onto Novex non-denaturing 6% gels and run the gel in 0.5X or 0.25X TBE for 1.5-2hours at RT. Then I use a gel analyzer to get an image of the gel.

I see that 80-90% of the probe is bound with the addition of nuclear extract, but the bands are at the very top of the gel wells, almost as if the protein-DNA mix is not entering the gel at all. This binding seems to be specific as addition of 25X-50X of the cold probe competes away all of the band at the top, and a non-specific competitor does not. Can anyone please tell me if this is normal, and what I can do to improve it? Am I using too much protein, which is causing protein-DNA aggregates or is it possible my PCR fragments are too big. Irrespective of what conditions I change, this pattern remains unchanged with small differences in intensity of the bands at the top. I would greatly appreciate it if somebody can help me with this. Thanks!!

Sowmya.
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Old 12-07-2011, 05:14 PM
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Default Re: specific protein-DNA complex staying up in wells in EMSA

You should titrate the amount of nuclear extract that you use. 10ug seems like a lot, but it of course depends how abundant your DNA-binding protein is. If you have saturated the probe at 10ug you can certainly try using less.

I think the size of your DNA probe is probably fine (I think people say about 250 bp is the practical limit for an EMSA probe, I typically use 100bp probes). The size of your probe probably won't have a massive effect on the mobility of the DNA:Protein complex. If the protein complex that binds to your DNA probe is very large it may not migrate into the gel very easily.

If your complex does not migrate into a polyacrylamide gel, you could try performing the EMSA using a TBE agarose gel. Also you could use a lower acrylamide concentration (try 4.5% or 5%).

Just out of curiosity is it normal to use 0.5uM probe with the non-radiactive EMSA systems? I have always just used 32P for my EMSAs and I add a really tiny amount (roughly 50 picoMolar).
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complex , dna emsa , emsa , fam labeled probes , nuclear extract , pcr probes , protein-dna aggregates , proteindna , specific , staying , wells


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