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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

EMSA problem - Electrophoretic Mobility Shift Assay Forum

EMSA problem - 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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  #1  
Old 10-27-2008, 09:37 AM
Pipette Filler
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Default EMSA problem



Hi all!

I'm using the EMSA Kit from Pierce to carry out my DNA-EMSA experiments.

I've been consistently getting 2 rows of 'white spots' in my gel photo.
Has anyone encountered this problem before? Is it because I'm adding too much protein?
I cant post the photo here yet as I'm new to the forum.

Thanks for your help!
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Old 10-27-2008, 03:14 PM
Pipette Filler
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Default Re: EMSA problem

Oh, I've another newbie question to add.

Is there a need to change new 0.5x TBE buffer after pre-running for the actual sample run?

Is it ok if I re-use the same buffer which I used for the pre-run?
Or should I prepare a new stock of TBE?

Is it necessary to do the pre-running and actual running in cold conditions?

Thanks!
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Old 10-29-2008, 01:40 PM
Pipette Filler
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Default Re: EMSA problem

I was told by Pierce that you can reuse the buffer but you need to remix it after the prerun because all of the solutes will run to the lower chamber. For your two rows of white spots, is this at the level of the shift or unshifted DNA. I posted just before you in this column about how I get a "double-band" in all 3 lanes at the level of the shift. If it is at the level of the unshifted DNA it could possibly be single and double stranded DNA.
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