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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-complex trapped in well

EMSA-complex trapped in well - Electrophoretic Mobility Shift Assay Forum

EMSA-complex trapped in well - 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 08-08-2011, 09:23 AM
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Default EMSA-complex trapped in well



Hi, I'm using EMSA to validate the binding ability of protein to RNA and also trying to get the binding sites from RNA. I've synthesized short biotinylated RNA and detection of shift using Streptavidin-AP after blotting onto nitrocellulose membrane. I separated the complexes on 2% agarose gel, shifts were observed but not very well separated, so i changed to separation on PAGE, initially with 8% PAGE, but all complexes were stuck on the well even the RNA itself (negative control), then i changed to 4% PAGE. However, similar incident happened and i do not get any shift as on agarose gel. I have no idea why the complexes and even RNA were unable to migrate down from the PAGE well eventhough the percentage was rather low. Hope someone can provide some insight into this issue. Thank you
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Old 09-02-2011, 05:49 AM
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Default Re: EMSA-complex trapped in well

I've managed to figure out the reason. The protein:RNA complexes were too big that can only be separated on agarose gel due to the pore sizes larger than in PAGE.
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Old 09-28-2011, 11:11 AM
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Default Re: EMSA-complex trapped in well

Hi Jenn, currently you are only using agarose? I found something that maybe able to help you.

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