-Amounts below are for 2 IPs per antibody
-Volume is for settled beads not for slurry
1. Equilibrate ~110 µl Biorad AffiPrep Protein A beads into PBST (PBS + 0.1% Tween-20) in an eppendorf tube. Wash 3-4 x 1 ml; use minifuge to pellet between washes.
2. Resuspend beads in 500 µl PBST and add 55 µg of affinity-purified antibody (use random rabbit IgG as a control). Mix for 30 min. - 1hr at RT.
3. Wash beads 3x with 1 ml PBST.
4. Wash beads 3x with 1 ml 0.2M sodium borate, pH 9.0 (dilute from a stock of 1M sodium borate, pH 9.0; make the stock from boric acid by pHing with NaOH). After the final wash, add 900 µl of the 0.2M sodium borate, pH 9 to bring the final volume to ~ 1ml.
5. Add 100 µl of 220 mM dimethylpimelimidate (DMP; 20 mM final; Sigma D 8388; FW 259.2; stored in a dessicated box at –20° C). Rotate tubes gently at RT for 30 min.
To make the DMP: Let bottle sit tightly closed at RT for 20 min before opening. Weigh out
DMP and leave dry until just before use. Resuspend in appropriate volume of 0.2M sodium borate, pH 9 and add immediately to the bead suspension (e.g. for 34 mg DMP add 596 µl sodium borate).
6. After incubation with DMP, wash beads 2X with 1 ml 0.2M ethanolamine, 0.2M NaCl pH
8.5 to inactivate the residual crosslinker. Resuspend in 1ml of the same buffer and rotate for
1h at RT. Resuspend beads in 500 µl. You can leave the beads in 0.2 M ethanolamine, 0.2M
NaCl pH 8.5 at 4° C until use. Beads are stable for several months at 4° C.
Per IP, use 50 µl (i.e. half) of the beads generated as per above.
1. Weigh out frozen cell material that has been ground with a mortar and pestal or a Warring blender 4-5 x in liquid N
2
.
2. Add an equal volume of Lysis Buffer (plus protease inhibitors) – see below
3. Setup sonication ice-water bath and sonicate
- 30% amplitude for 3 min total (15 s on; 45 s off - after each 1 min wait ~2 min to chill)
- 40% amplitude for 30s (15 s on; 45 s off)

(Save a CRUDE sample)
4. Transfer crude extract to TLA100.3 tube and spin at 20,000g for 10 min at 2° C, DECEL = 5
(Save a LSS sample)
5. Remove sup and pellet at 50K for 20 min at 2° C. Try to avoid any lipid and repellet if too cloudy.
(Save a HSS sample)
6. Collect sup into a tube on ice. Add KCl to a final concentration of 300 mM.
1. Pre-elute 50 µl of coupled beads 3X with 1 ml of 100 mM glycine pH 2.5; this gets rid of uncoupled/elutable antibodies and reduces background. Do this quickly – don’t leave beads for long time in glycine.
2. Wash beads 3 x 1ml into Lysis Buffer (with 300 mM KCl) to neutralize glycine.
3. Mix beads with 900 µl HSS extract for 1 hr at 4° C.
4. Rinse beads 3X with 1 ml Lysis Buffer containing 300 mM KCl, 0.05% NP-40, 0.5 mM DTT, and protease inhibitors (1:1000 LPC; 1:2000 E-64)
5. Wash beads 2 x 5 min with 1 ml Lysis Buffer containing 300 mM KCl, 0.05% NP-40, 0.5 mM
DTT, and protease inhibitors (1:1000 LPC; 1:2000 E-64)
6. Wash 1X with Lysis Buffer with 100 mM KCl without detergent. Remove as much supernatant as possible.
Sample Buffer Elution:
1. Elute beads by heating in 50 µl of 2X SB WITHOUT DTT for 10 min. at 50° C.
2. Pellet beads, transfer sup to a new tube and add DTT to 100 mM (1/9 supe vol of 1M stock;
ELUTION 1).
3. Add 50 µl 2X SB WITH DTT to pelleted beads (ELUTION 2).

4. Boil both elution samples for 5 min and analyze by silver staining/blots. Generally, you will get something in both elutions although amounts in each are variable; ELUTION 2 will have more IgG contamination than ELUTION 1.
5. For silver staining, load 5-10 µl directly. For blots, load same amount of a 1/10 dilution made in SB.
Urea Elution:
1. Wash beads with pre-urea wash buffer (50 mM Tris pH 8.5, 1 mM EGTA, 75 mM KCl).
Remove all residual supernatant
2. Add 75 µl Urea elution buffer (50 mM Tris pH 8.5, 8 M Urea – For urea, use Invitrogen cat
# 15505-035) and rotate for 30 min at RT.
3. Pellet beads and remove urea to a new tube. Repellet to ensure removal of all protein A beads.
4. Remove 50 µl of urea and drop freeze in liquid N
2
to send for mass spec. Take the rest of the urea and add 3 x sample buffer to run on a gel.
Glycine Elution:
1. After standard IP, elute beads 3x with 150 µl 0.1 M Glycine pH 2.6. Pool elutions and neutralize by adding 150 µl 2 M Tris pH 8.5. Neutralize the beads by washing 2x with 150 µl
Lysis Buffer (without detergent) and pool with eluate. Total volume will be 900 µl.
2. Add 1/5 th volume 100% TCA (~200 µl). To make 100% Trichloroacetic acid (TCA) take 125 g and add 56.75 ml H
2
O.
3. Leave samples on ice overnight in coldroom.
4. Spin for 30 min at top speed in microfuge at 4°C. Aspirate the sup with a gel loading tip leaving 5-10µl so as not to disturb the pellet.
5. Wash 2 x 500 µl of cold acetone. Spin for 10 min. after each wash.
6. Speed vacuum for 5 min.
6. Can send frozen tube directly for mass spec.

6. If want to run on a gel, resuspend pellet in 40 µl of 1x sample buffer with 0.8 µl 1 M Tris
Base. Heat sample for 5 min vortexing intermittently.
50 mM HEPES, pH 7.4
1 mM EGTA
1 mM MgCl2
100 mM KCl
10% glycerol
0.05% NP-40
Just prior to use, to 5 ml lysis buffer, add 1 tablet mini EDTA-free Complete PI tablet