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Immunoprecipitation of protein complexes - My preciousss protein complex (Mar/01/2006 )


I have a question. I am trying to precipitate some protein complex. I know that the protein isolation step is crucial for the complexes not to fall apart.
Therefore I got rid of SDS in the lysis buffer, not to denaturate proteins, and I used Triton instead. Are there any other ingredients I shouldn't use in my lysis buffer? I start from modified RIPA and I can't afford any kits. Do you maybe use specific buffers when you want to IP a whole complex?



The basic ingredients I use are:

a buffer, usually Tris at like 100mM pH 7.4

Isotonic saline or ~150mM

0.2%-1.0% of your favorite detergent
Personally I use NP40 as it seems to be a milder detergent, which can be important to preserve interactions.

A Phosphatase inhibitor, as some interactions can be phos. dependent.
Sodium Orthovanadate is an economical option
as is Sodium Fluoride

Protease inhibitors:
We use a Complete mini tablet from Roche which is a mixture of like 20 different protease inhibitors easy but a bit expensive. They are like $200 US for only enough to reconstitute 250mL of lysis buffer.

If those are too expensive, I've seen people use just leupeptin, pepstatin, and PMSF (a chemical), as a protease inhibiting cocktail, in which you make your own solutions though.

I hope this is helpful



QUOTE (Mountainman @ Mar 2 2006, 04:21 AM)

The basic ingredients I use are:

Yes, it seems we're using the same buffers, and I make the inhibitor cocktail you mentioned. Only there's SDS in the literature, too.
Because SDS is denaturing proteins, I added triton instead. So you say NP40 alone is enough? I thought it will be too mild to break the nuclear membrane.


Not sure about NP40 and nuclear membranes. I use it to solubilize and IP a plasma membrane protein and it seems to work for that ok.

I've seen published IP buffers with Triton X although somewhat less often. You can always try both I suppose depending on what you are doing.