Rna Emsa

[tara29466]

Hello all,
I am researching the RNA EMSA technique. I currently have cDNA constructs that I want to test for protein interactions, the one I want to test is ~300 bp. Is this short enough for EMSA??
Thanks,
Tara

[moleculardude]

Hello Tara welcome to the forum!

I have done a few RNA emsa's... are you testing for RNA splicing or UTR untranslated region (5' or 3' UTRs ?) - translation interactions ?

your cDNA may or may not have the RNA spicing or UTR areas ... please mention which area you will study....

300 bp is fine for now, you must clone it into a T7 / T3 construct and express it as an RNA... you must then take cytosol and add it to the RNA in proper buffer (salt) conditions.

Eventually you will need to make smaller probes to find a more specific area of interaction. You might also try non-radioactive EMSA. Pierce makes a good Light Shift Chemiluminescent kit which can be used for RNA EMSA as well as DNA.

please respond for those questions and we can help more!

[tara29466]

Hi-thanks for your quick reply I am looking for both 5' UTR regions for protein interactions. Although, interesting that you ask, we are checking those regions for regulation of splicing. Right now from deletion constructs, I have narrowed one area of regulation to about 300 bp, just wanted to know how much further I need to go
Tara

[moleculardude]

we are here to help! maybe I can get your help in the future...

300 bp is quite big although not bad... problem is there may be MANY proteins binding to this region...

you could try UV-crosslinking / denaturing EMSA (for RNA-interacting proteins)


and also non-denaturing EMSA in native gel (for protein complexes)

Also I suggest you use smaller probes ie 50 nt (all you need are 6 constructs - may be a lot but definitely worth it to publish a good paper - 6 X 50 covers your 300bp fragment) as you will want to publish (eventually) where these proteins are binding (general region)...

[jamsheer]

hi everyone,
I'm working on plant proteomics and I'm going to do RNA-EMSA with my protein. in silico analysis shows that the mammalian homologues of my protein bind to RNA non-specifically. So, firstly I need to confirm it. I want to know that is there is any type of RNA which is routinely used for this purpose (like ΦX174 RF I and ΦX174 virion DNA used for DNA-EMSA)...I want both ss and dsRNA.....
thanks..
jamsheer