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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-strange result

EMSA-strange result - Electrophoretic Mobility Shift Assay Forum

EMSA-strange result - 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 08-08-2008, 10:40 PM
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Default EMSA-strange result



Hi There!

I am working on transcription factor characterization. I am using biotynalated, PCR-amplified promoter region and His-Tagged protein which is purified by using affinity chromatography. I have clearly observed the DNA: protein interaction by EMSA and that part is okay.

When I used chemicals /effectors/ligands to check the effect , one of the ligands showed strange result I got a shift band and no unbound DNA (as seen from the control) at highest ligand concentration (1mM) whereas at 0.5mM,0.25mM and 0.01mM there was binding and I could see both shift and unbound DNA.
Next time when I used the same protein (now a day old stored at 4 deg) and carried out EMSA with the 1mM ligand there was no shift but a control whereas at other concentrations result was consistent.

When I used new protein aliquot from -80 deg I got same shift but no unbound DNA at 1mM.

Can anybody tell the possible reason?

Is it the biotynalated promoter part getting masked after DNA: protein: effector interaction so that there is no super shift signal but just a shift because of DNA: protein interaction since this is the just DNA: protein and no effector is available since it is already complexed but the DNA is not visible since its biotynalated part is masked because of the interaction?
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Old 09-04-2008, 10:30 PM
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Default Re: EMSA-strange result

hi this is interesting

Can you post a gel image so we can analyze this further?
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