immunoprecipitation: beads-antibody incubation - (May/18/2010 )
my question is about a changement of one step in my protocol of immunoprecipitation IP. which is the incubation of antibody with beads protein G, that i performed, before crosslinking and adding cell lysate, on rotation at 4°C. i could immunoprecipitate my protein of interest.
previously, i have performed IP by incubating antibody with beads at room temperature RT on rotating wheel.
so, im wondering if it is worths to keep 4°C or room temperature??
in dynabeads prot.G datasheet, the incubation is at RT, but, since i read in another protocol, using the same lysis buffer as mine (RIPA), they incubate antibody with beads at 4°C, before crossling step with DMP.
If you have any idea about this, please help!! i dont know what to keep (4°C or RT)
since the dynabeads data sheet says to incubate at room temperature (and it works for you), i would not change.
if you want to change then you will have to determine whether it works or not or requires longer incubation by trying it yourself.
after all that is said, it will probably work at 4C with a longer incubation (but i still recommend that you don't change).
ok, thank you for the advice.
i was also thinking in my next IP to keep RT incubation, and see.
The incubation temperature influences the binding kinetics of the immunoprecipitation. Binding occurs faster at room temperature, and as the incubation period needed is so short, there is no reason to perform the incubations at 4 degrees. At room temperature, it is enough to incubate for 10 min both for the beads and antibody incubation and for the target capture.
If you decide to incubate at 4 degrees you should increase the incubation time to 30-60 min, this is especially important in the target capture step. If you want to do an over night incubation we recommend to do that at 4 degrees.