Immunoprecipitations 3/02 Beth
Day 1
1) Prepare beads (protein-A agarose- Gibco BRL life tech) take 500uL and place into 3mLs of cold PBS, invert several times. Spin in clinical centrifuge to pellet beads for 3 minutes setting 3. Do NOT spin too fast or the beads will break and you will get lots of non-specific binding to them etc.
2) Wash 2 more times in 3mLs PBS and resuspend again in 3mLs and divide this suspension into two tubes, one is your control/preincubation set of beads, the other will be bound to your antibodies. To bind your antibodies, you will have to empirically determine how much you need to add but a good start is 1mL of an affinity purified preparation of antibody that you use on a western at 1:1000 or so. Each mL of ProteinA beads binds 20mg IgG. Rock the beads with antibodies O/N (you may be able to do this less time but the IP itself takes a long time with all the washes so I like to just set this up ahead of time.
Day 2
3) 45 minutes prior to completion of preparation of the high speed supe or extract (HSS derived from brain 2g~2mLs), wash the antibody bound beads to get rid of any non-bound antibodies. To do this pellet the beads, remove supe that contains if any antibodies unbound. Add 3-5mLs PBS and resuspend beads by inverting the tube, then pellet the beads and repeat this 2 times. On the last spin, decant the supe wash and leave only a little buffer on top so that the beads do not dry out.
4) Take a gel sample of the HSS or extract. This is your load. Add the HSS to the beads you made yesterday that do NOT have ANY antibodies on it, this will serve as a pre-blocking resin to bind up any non-specific binders. It will also serve as a control that you wash and elute in parallel with the antibody bound protein A beads. Incubate the HSS/extract with the control beads for 1/2 hour at 4°C with shaking. You may want to also try the IPs at room temperature.
5) After the half hour, spin the control beads to collect supe. Place the Supe onto the antibody bound beads and put PBS onto the control beads. Incubate the HSS/antibody beads at 4°C for 3 hours with shaking. In the meantime wash all HSS from the control beads by washing the beads in PBS 3 times.
6) After 3 hours, remove HSS (take a gel sample- this is you flow through). Wash both the control and antibody bound beads in parallel from this point on. Wash the beads in PBS or lower salt buffer, until you are certain of an interaction not being disrupted by higher salt, keep taking gel samples of the washes to see if any weaker but specific interactions are occurring. Wash 3 times with this buffer. You can even divide the beads into 4 tubes and try 4 different stringency washes.
7) Wash the rest or most rigorous wash using 0.5X or 1X RIPA (you can also try this without deoxycholate if you are having trouble with an interaction getting washed out.)
8) Wash 3 quick changes in the RIPA and then spin the beads a final time. When you are done with all washes (which as I mention you should determine empirically) remove all extra liquid from the control and antibody bound beads.
9) Per 500uL of beads, add 15uL of 5X lamelli sample buffer and mix until all the beads have come into contact with the 5X sample buffer. Boil for 5 minutes. Cool for a few minutes with the eppendorf inverted, while cooling, label a new set of tubes. Relieve any pressure from the sample by opening the lid, shut the lid and Then using the smallest gauge needle 26, poke a hole into the bottom of the inverted tube, and place the bottom of the tube into the labeled eppendorf that is now your receiving tube. Spin at 6000 rpm for 10 seconds (not too fast or the beads come through the hole and all you want is the sample). The beads will now by a light blue.
50mM Tris-HCL pH 7.5
150mM NaCl
1% Nonidet P-40 detergent
0.5% deoxycholate (powder 414.6)
MODIFIED RIPA- try without deoxycholate and range the nonidet to 0.5%