Immunoprecipitation (IP) washes - (Dec/15/2017 )
Lab debate: Do you need to rotate your samples during the wash steps for an IP? There seems to be an unwritten rule somewhere that you add your wash buffer, rotate for 5 minutes at 4 degrees, spin down, and repeat. Is that 5 minute rotation standard/necessary?
It depends on how stringent you want the washes to be. The longer the wash, the more stringent it is/the greater the chance of loosing bound antibody is. Personally I tend to resuspend the beads by gentle rocking for a minute or two then repeat the wash a couple of times.
We have a protocol with 4 washes, and I've seen general recommendation to mix by gentle inverting few times.
I can't say I see any differences, from times when I didn't inverted them because noone told me yet. And since the time I do it, rather same I would say, I did have also few cases that looked not well washed.