lysis buffer preserving protein-RNA interactions

[Lechu]

On Mar 2, 4:47 pm, "Pow Joshi" <pow.jo... from gmail.com> wrote:
> On 2 Mar 2007 04:10:56 -0800, Lechu <lech_kaczmarc... from yahoo.com> wrote:
>
> > Hi all,
>
> > Could anyone recommend me some lysis buffer for brain tissue,
> > preserving RNA-PROTEIN interactions. I plan to do mRNA 'pull-downs'
> > with rec HisTagged RNA binding protein. Want to use both, isolated
> > mRNA and crude cell lysate.
> > HEPES-based ELB buffer will do? Any other suggestions?
>
> ah, I did some protein pull down assays with the brain tissue ....
> works well with the Hepes +NP-40/TritonX +protease inhibitors and
> EDTA.... I suppose you would have to add some DTT/DEPC and Rnase
> inhibitors, to preserve your RNA ......

To prevent degradation I use RNAsin from Promega (RNAses inhibitor) +
DEPC water. Will consider DTT. The problem is that the bait protein
tends to clump (aggregate), unless in 2M urea. RNA bindig buffers
working for EMSA assays contains urea, the question is how to get the
buffer buffer allowing for: lysing of the tissue without altering RNA
binding (this should be still pretty easy) and without making the
protein precipitate (that's the challenge).

Buffers are as follows:

RNA binding buffer (used for gel-shifts):

10 mM Na-HEPES (pH 7.6)

100 mM KCl

1-3 mM MgCl2

0.1 mM CaCl2

5% Glycerol

DTT 1-5 mM

0.1 mg/ml BSA

optionally 100uM Zn2+



The BAIT protein was dialyzed against a buffer containing 2 M urea,
100 mM KCl, 10 mM Na-HEPES (7.6), 1 mM DTT, 0.1 mM CaCl2, 1 mM MgCl2
and 5% glycerol. This is how the protein was prepared for RNA binding.


W/o denaturants the protein makes aggregates on the NiNTA beads.


Thanks for any suggestions.