co-IP, temperature ?
peter_loesh at hotmail.com
Sat Aug 26 06:35:54 EST 2000
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,
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