immunoprecipitation: beads-antibody incubation
Posted 18 May 2010 - 01:41 PM
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)
Posted 19 May 2010 - 06:03 AM
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).
genius does what it must
i do what i get paid to do
Posted 20 May 2010 - 02:49 AM
i was also thinking in my next IP to keep RT incubation, and see.
Posted 30 June 2010 - 02:10 AM
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.