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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.


Maximum Oligonucleotide Size and Gel Percentage

Maximum Oligonucleotide Size and Gel Percentage - Electrophoretic Mobility Shift Assay Forum

Maximum Oligonucleotide Size and Gel Percentage - 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 08-29-2008, 02:40 PM
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Default Maximum Oligonucleotide Size and Gel Percentage



Hi. I am just starting EMSAs for the first time. My promoter sequence is 2.5 kb, and I tried to perform EMSA with this sequence. I found that my probe alone without any nuclear extract did not even enter the gel when I used a 6% gel. Does anyone know the maximum size of an oligo that can be used with EMSA. I realize that such a long sequence would bring many nuclear proteins with it and would possibly make the band very fuzzy, but is it possible to use a longer probe with EMSA? If so, what percentage gel and what conditions are favorable for this? Thanks.
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Old 09-04-2008, 10:36 PM
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Default Re: Maximum Oligonucleotide Size and Gel Percentage

Hello,
wow that is tough its quite big. Why are you using this 2.5kb promoter sequence? You will need a smaller gel percentage and even possibly agarose gel.

Can you isolate a smaller binding element from the 2.5kb promoter using Matinspector or similar promoter analysis tool.

The problem is if you use non-denaturing gels with a 2.5kb promoter fragments dozens or hundreds of proteins would be binding this 2.5kb fragment and its unlikely to enter the gel.

I would use a much smaller fragment (even 200bp), to analyze this. If you really need to analyze a large fragment use a different method such as a DNAse protection assay.
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