co-IP, temperature ?

[Peter Loesh]

Hello to wizards !

Can somebody help me to choose correct conditions for a 
co-immunoprecipitation experiment ?

my case is:

I use different buffers/temperatures for imminoprecipitation:

1) 1% NP40 in PBS(400mM NaCl, to get nuclear proteins off DNA)
2) RIPA (1% NP40, 1%NaDOC, 0.1% SDS in PBS)(just 150mM salt)
3) temperatures: 2C, 12C, 25C.

(buffers contain also protease and phosphotase inhibitors, iodoacetamide and 
EDTA). Lysates (mammalian cells) are passed few times through a needle to 
reduce viscosity. Phosphate buffer is at pH7.

it seems that 1% NP40 is the most stringent buffer !
and the higher the temperature, the more additional bands I get !
Thus, when I use RIPA at room temperature I get just all co-precipitated 
together with my protein.
Is this normal ????

I thought that at higher temperature stringency would always be higher...

(My idea is that with RIPA I get solubilization of more proteins, and may be 
partial denaturation because of SDS (?))

specific question is:
1) what is more stringent RIPA or 1% NP40+salt ?
2) is higher stringency expected at higher temperature during IP ?

hope someone will explain me this,
thanks in advance,

Peter



Peter
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