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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.


Native gel or Agarose for RNA EMSA?

Native gel or Agarose for RNA EMSA? - Electrophoretic Mobility Shift Assay Forum

Native gel or Agarose for RNA EMSA? - 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 11-30-2011, 06:11 AM
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Question Native gel or Agarose for RNA EMSA?



Hi there,

I am going to do a biotinylated RNA EMSA. I would like to know what is the difference and how does it matter if I go for a native gel or an agarose gel.

Also, do you guys make individual components of a buffer (EDTA, DTT, APS) in DEPC treated water?
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Old 11-30-2011, 06:29 AM
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Default Re: Native gel or Agarose for RNA EMSA?

What percentage gel shall I make? My RNA is 2.4kb and protein is 68kD
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Old 11-30-2011, 04:24 PM
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Default Re: Native gel or Agarose for RNA EMSA?

Quote:
Originally Posted by Identity_crisis View Post
Hi there,

I am going to do a biotinylated RNA EMSA. I would like to know what is the difference and how does it matter if I go for a native gel or an agarose gel.

Also, do you guys make individual components of a buffer (EDTA, DTT, APS) in DEPC treated water?

For EMSA you need to run either a native polyacrylamide or a native agarose gel. People often use 0.5x TBE as a buffer.

It's probably not absolutely necessary to use DEPC water, Milli-Q should be sufficiently pure (assuming your glassware isn't contaminated w/ RNaseA).

I think you will have trouble detecting a mobility shift for a 2.4 kb RNA on a polyacrylamide gel, so you may need to go for an agarose gel. The alternative is to use a fragment of the RNA that you think contains the binding site.
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