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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.


EMSA - Problem with gel interpretation

EMSA - Problem with gel interpretation - Electrophoretic Mobility Shift Assay Forum

EMSA - Problem with gel interpretation - 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 09-01-2010, 03:30 PM
Pipette Filler
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Default EMSA - Problem with gel interpretation



Hi. Couple weeks ago I started EMSA. My RNA is 260nt long and the protein molecular weight is 65 kDa. I attached the gel and I need Your help with interpretation. First I separate my samples on 8% gel (40:1) - left panel and recently I separated samples on 6% gel (75:1)- right panel. As You can see I obtained different results. I marked free RNA and probably the RNA-protein complex but can anyone explain me what is the band marked with ,,?''. I dont think so that is a complex with protein because is to close to free RNA and the protein is quite big. So what is it then?

The gel is a native PAA (50mM Tris/ 50mM glycine at ph 8.3)

Is it possible that band marked with ,,?'' comigrate with the RNA-protein complex on 8% gel because of the 40:1 ratio of acrylamide/bisacrylamide?

Any ideas?

Thanks in advance


blaszcz
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Last edited by blaszcz; 09-02-2010 at 07:27 AM.
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Old 11-22-2010, 07:56 PM
Pipette Filler
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Default Re: EMSA - Problem with gel interpretation

Hi, the only explanation that I can figure out is a different secondary structure of your free RNA that comes out just on 6% gel (75:1).

All the best

Mingu
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