Immunoprecipitation overnite or for several hours?

[oBWhat]

Hey guys,
should I immunoprecipitate overnite or for several hours?

thank u

[jpap]

It depends on several variables, quality of the antibody, affinity and specificity, stringency of the incubation buffer, abundance of your protein, to name a few.
If this is the first time you use this method I would recommend overnight incubation at 4C, and if the signal is strong you could try cutting down the incubation times.

[braveshirely]

generally, IP for 4 hours is good enough.

[danfive]

ON is safe if your antibody is specific and degradation is controlled via 4C or Proteinase inhibition. If your prep is clean and not a biological fluid you should be fine with ON. Biological fluids have endogenous antibodies that will bind to protein A/G and co elute with your eluant--learned this the hard way.