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-   -   EMSA RNA no shift (http://www.molecularstation.com/forum/electrophoretic-mobility-shift-assay-forum/3821-emsa-rna-no-shift.html)

andrealiliam 08-25-2008 03:15 PM

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.... :(

admin 08-25-2008 09:33 PM

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 :)

andrealiliam 08-26-2008 02:08 PM

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:notworthy:

Andrea


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