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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 RNA no shift

EMSA RNA no shift - Electrophoretic Mobility Shift Assay Forum

EMSA RNA no shift - 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 08-25-2008, 03:15 PM
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Unhappy EMSA RNA no shift



Hi i need a help

My english is not good but i tried....

I trying to make a EMSA RNA, but i dont have any shift... my RNA have 311nt... i have only my rna in the botton or the gel and in the top at the wells i have a little signal....

Please help me....
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  #2  
Old 08-25-2008, 09:33 PM
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Default Re: EMSA RNA no shift

Hello Andrealiliam,

its ok at least you speak 2 languages. I will try to help you. RNA EMSA no shift issues could be several things including:

- Salt (too much / too little) in the Buffer
- Inappropriate pH
- RNA inappropriately made (the binding area for the protein is not there)
- Binding-protein low abundance due to bad protein preparation (degradation of protein? not enough protein?)

Also is this a UV-crosslinking EMSA with denaturing ie b-mercaptoethanol and SDS or is this native non-denaturing EMSA?


Have you tried to optimize your shift with different buffers ie HEPES or Tris? What about different pH conditions? Different salt conditions? MgCl2? Yes there is a lot of optimizing for RNA EMSA Its probably the toughest of the experiments - its an art .

Please let us know also feel free to post your image by (Go Advanced) then go to add Attachments.

Cheers
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Old 08-26-2008, 02:08 PM
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Default Re: EMSA RNA no shift

Thanks admin...

It's a native non-denaturing EMSA. I purified my protein with a his tag as native conformation.. I tested alredy HEPES and Tris at pH 7.4 and 7.9 in the buffer binding and I dont have any results.. Also I tried MgCl2 at 2mM final concentration and nothing..... Now I am making a new protein maybe during store at -80C my protein was denatured???...

I tell you if Ihave some results.....

Again Thanks a lot

Andrea
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