CoIp RNA

[Jorge]

Hi,

My name is Jorge, I am trying to do some Coimmunoprecipitations of RNA with tagged-proteins. I am using the Ribonucleoside Vanadyl Complex, and in my hands it makes to precipitate RNA unespecifically. So, I have really nice CoIps, included the negative controls :-(, in which I obtained a High background due to this problem.
Anybody has this problem?
Anybody knows what is happening? or, what I can do?

Thanks in advance,

Jorge

[koukou2]

Hi Jorge!

I assume you are trying to IP the proteins and then assess the RNA....

there are some problems with this if you do not wash well there could be RNA in your IP...

also how are you IPing? could your immunoprecipitin have bacterial RNAs? are you preparing this from bacteria or buying it?
even if you have tagged proteins you are obviously using some kind of immunoprecipitin... you should buy this or maybe use beads to diminish the posiibility of contaminating RNAs in the immunoprecipitin

[koukou2]

also are you crosslinking?


another thing....

have you done RNA-EMSA before with radiolabeled or biotinylated RNA?
as this is the standard method for assessing protein-RNA interactions...

[Jorge]

Thanks Koukou2 for help me,

Yes I IP the proteins. I see them by Western blot and i do not detect background at all.
The washes? I wash 5 times 5 minutes and 50 volumes each in a rotating wheel.
My RNAs are from yeast and the cultives are chekced. I asses the RNA in Northern blot with specific probes. So I could say I have not contaminated RNA.
I have tried several times with:

Antibody plus gammabind sepharose

Antibody-sepharose

in both cases I have this unespecific precipitation.

The buffer is prepared with the Ribonucleoside Vanadyl Comples at 52ºC to dissolve it completely and then filtered in 0.22microm.
But during the precipitation, often I have some lumps that I think is what makes the trouble.
I am sure that avoiding this lumps I will avoid the unespecific precipitation, but I do not how.
And not I have not make RNA-EMSA, because I want to know wich rRNAs bind to a protein in different conditions.

I wonder if anybody have use this RNAse inhibitor and could tell me how to use it, I would be very grateful.

[Topaz2]

Hi Jorge,
which antibody are you using to IP? is it polyclonal or monoclonal and from the same species?

[Jorge]

Hi Topaz2,

I am using an anti-MYC monoclonal antibody, because my proteins are MYC-tagged.

[Topaz2]

hmm very difficult....i need to think about this