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Emsa

Emsa - Biochemistry Forum

Emsa - Discuss and post questions regarding the study of Biochemistry. If you need homework help this is the place to ask!


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Old 04-23-2013, 06:34 PM
Pipette Filler
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I am trying to monitor the activity of a DNA binding protein for the first time by electrophoretic mobility shift assay (EMSA) and have a couple questions. First of all I am monitoring a small protein (10.5kDa) binding to a short oligo (possibly in monomeric and/or dimeric fashion). I read that most EMSAs are done using 6-8% polyacrylamide gels, will this be enough to resolve my bands (I usually use 15% to resolve such bands on SDS gels)? Also why do most protocols call for no stacking gel, is there harm in using a stacking gel? Thanks.

-ICS
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Old 04-24-2013, 08:20 PM
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In my experience, a 6% polyacrylamide (PA) gel resolves bands above 100 bp, 8% might resolve a little further. I have only gone to a 12% PA gel, which resolved me bands from 70 to 40 bp quite well, the latter showing a bit faint, though.

EMSA technique allows you to see if a protein of interest binds to DNA, this is done by using a probe (radio/fluorescent-labeled DNA) containing the putative binding sequence for the protein. After hybridize probes with proteins, inhibitors or whatever your essay requires; you run the probes in PAGE. Free (unbound) probes will show an electrophoretic pattern while protein-bound probes will be "delayed", this is, they will migrate less across the gel than unbound probes.

The above paragraph is to show you that in EMSA you run DNA so it requires a DNA PAGE not a protein PAGE (SDS-PAGE). DNA does not need to "stack" as it is done for proteins, so PA gels are done in one piece and they run smoothly, just look at the picture.

Hope this helps you.

By the way, the pic I post is a PA gel with silver staining showing a BSA (protein) band (upper), DNA fragments (below) and DNA ladder (right).
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Old 04-25-2013, 02:51 PM
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Default Re: Emsa

luisillo,

Thanks for the help, we have our probes radiolabeled (which are only 23bp btw) and are ready to go. I think I'm going to start off with a 10% PA gel based on suggestions from other colleagues, and only move up to 12% if I can't get the resolution I need. Also, it makes sense that since we're monitoring DNA size there isn't a need for the stacking gel. Thanks again!

-ICS
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