co-IP, temperature ?
martin.offterdinger at akh-wien.ac.at
Mon Aug 28 02:21:22 EST 2000
"Peter Loesh" <peter_loesh at hotmail.com> schrieb in im Newsbeitrag:
F242OAmEaJswFCHOzyf000000a3 at hotmail.com...
> 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
> 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
> 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,
If you get more bands at higher temperatures this could also be a
degradation problem---> higher temp----> higher activity of proteases. I
think that this explanation is more probable than yours, so be very careful!
I never heard about IP at elevated temp.; normally you should do it at 4°C
not higher or lower (for the above mentioned reasons) and just play around
with detergents, salt, etc..
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