I'm new in proteomics and doing IP with A/G agarose beads, but I struggle with what is the best way to pipette them.
Here they don't resuspend the whole volume, just a bit and semi-randomly aspire 30 ul of partly mixed beads. This leads to uneven volume of beads in each sample and it's sometimes very difficult to pipette. And since I just remove all except the beads at the end of wash and mix with fixed volume of Laemmli, this leads to uneven final volumes also (we do not measure protein concentration, just use it as it is, so then uneven protein concentration).
I was trying to find ways to improve this, I got some information that mild vortexing the beads alone (before adding to lysate) would not damage them and evenly mix the whole volume so I can pipette it more evenly. Well, mostly, sometimes there are very littel beads.. but bigger problem is, vortexed beads will attach on the well walls and they won't go down even by spinning.
I decided to check visually the volume of beads before incubating, but still I wonder, what is the best trick to handle those.
Incidentally, the protocol used here incubates sample with Ab for 1-2 hours rotating, and then overnight (in coldroom) with beads before washes. I was one one day short last time and needed to shorten it up and manufacturer bead protocol said two hours of incubation could do it, so I tried 2 hours with Ab alone, 2 hours with beads. This IP ended up badly (this is a repeat of an successful one, so it should work ) and I wonder if shortening the incubation could have the effect (or beads damage since I did vortex them to mix, they are not very eager to). I try not to disturb the complexes by rough mixing of course.
This problem may repeat, since I'm kind of forced to finish even those experiments where I'm a day behind. So my option is either to IP in one day (not making bead incubation overnigh) or leave it in the rotator for the whole weekend (can't go to lab). Which do you think would be better option? (I still don't know if the shortening of beads incubation caused the experiment to fail, but it could).
I would be grateful for any other tips regarding work with the beads, thanks.