Sfeir Lab, Aleks Penev, 8/21/14

Immunoprecipitation Protocol
1. Collect and pellet cells as for Western blot (500k cells, spin down at 1200
rpm)
2. Make Base IP Lysis Buffer:
(10ml)
Glycerol
10%
1ml
Tris-HCl (pH 7.4)
50 mM
500ul
NaCl
150mM
300ul
IGEPAL/Nonindet NP-40
.5%
50ul
Triton X-100
.5%
50ul
EDTA
1mM
20ul
ddH2O to fill
up to 10ml
To make Complete, add:
Roche Protease Inhibitor 1 tablet/10ml
PMSF (stock 100mM)
1mM
DTT (stock 1M)
1mM

1 tab
100ul
10ul

3. Keep all lysis buffers and protein samples on ice or in cold room.
4. Wash cell pellet in cold PBS 2x, then resuspend in Complete lysis buffer. Use
500ul per 50mg wet pellet. Approximately 1ml/confluent 10cm plate;
2ml/confluent 15cm plate.
5. Triturate mixture ~20 times with p200 pippette.
6. Incubate sample on ice for 30min.
7. Sonicate for 5min (10sec ON, 20sec OFF) using 15ml conical tube adaptors.
8. Spin down in eppendorf tubes @ max speed (15k rpm) for 10-15 min, 4C to
pellet cell debris.
9. (optional) Take supernatant and "pre-clear" with Protein A/G beads (10ul/ml of
sup.) to reduce background on IP. Incubate end-over-end for 1hr in coldroom
and spin down.
10.Save 5% of volume as input. Add 4x sample buffer, boil for 5min in sand,
sonicate/shear as for western then store in -80.
11.Take rest of the supernatant and add 1-2ug antibody per 1ml supernatant.
*If there is no empty vector control, it is necessary to split supernatant in half
and use half as IgG control (background binding to IgG constant region) and
the other as the sample.
12.Incubate mixture in cold-room on end-over-end rotator O/N.
13.Pre-equilibrate Protein A/G with lysis buffer wash for 20 min.
14.Add beads (10ul/mg Ab used) and incubate in cold-room on end-over-end
rotator for at least 2 hours (up to 6 is ideal).
15.Spin down beads @ 1200rpm for 30sec and allow to settle before aspirating.
16.Wash 3x 5min in incomplete lysis buffer, EOE rotation.
17.Wash 1x 5min EOE in cold room w/ incomplete buffer + low salt (150mM KCl),
1x 5min w/ incomplete buffer + high salt (300mM KCl) and 1x 5min with
Wash #4.
18.Spin down and resuspend in 20ul 1x PBS, then add 20ul 4x sample buffer.
19.Boil 5min on sand (95C). Can freeze in -80 or load immediately.

Sfeir Lab, Aleks Penev, 8/21/14

20.DO NOT LOAD INPUT NEXT TO IP SAMPLES - the input signal will blow out any
IP signal.