co-IP, temperature ?
rand106 at hotmail.com
Sat Aug 26 23:45:52 EST 2000
In article <F242OAmEaJswFCHOzyf000000a3 at hotmail.com>,
peter_loesh at hotmail.com ("Peter Loesh") wrote:
> 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,
I trust you experimental results. They are telling you what it is.
Years ago, when nonionic detergents were widely used to "reconstitute"
membrane proteins, people discovered that nonionic detergents become more
stringent when the concentration of salt is increased. And 400 mM salt was
the magic number they used.
This obviously had to do with the CMC (critical micellar concentration),
which in the case of NP40 is between 0.05-0.3 mM at 50 mM Na+. When you
increment the salt the CMC decreses. For membrane reconstitution a detergent
is a detergent above its CMC, simply because in that state it offers the 2
faces necessary for dispersion.
In RIPA you have NP40 and also have DOC (CMC=1.5 mM) and SDS (CMC=2.3 mM)
but the ionic strength is low.
Regarding the correlation between temperature and stringency I am not sure.
It may also be an effect on the CMC of the detergent. May be the thermal
energy tends to increase the detergent CMC making its "detergenticy" less
powerfull. Just an speculation.
If I were you I'll stick to the results provided they are reproducible. Let
the Biophysicist find out the molecular reasons for the "stringency"
variations you found.
One last thing: Detergents must not be regarded as real solutes. The action
of a detergent is always dependent on the ratio detergent/material to be
dispersed by it. In other words 0.1% Triton acting on 15 mg lipids (as
liposomes) is different than 0.1 % Triton acting on 0.1 mg lipids. The same
applies for extraction of crude cell lisates as in your case.
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