1. StaphA Buffer

Phosphate Buffer A                8 ml (13.9g NaH2PO4.H2O/500ml)
Phosphate Buffer B                42 ml (26.8g Na2HPO4.7H2O/500ml)
10% Triton                           100 ml
SDS                                     1g
NaN3                                   1g
4M NaCl                               25 ml
0.5M EGTA                          10 ml
H2O                                     815 ml
Sodium deoxycholate (RT) 0.5% prior to use.!

2. StaphA-Lysis Buffer: (Add phosphatase and protease inhibitor fresh)

1mM PMSF (MW:174.2, 100mM stock in ethanol (17.4mg/ml), -20~80^oC)
10ug/ml aprotinin (1 mg/ml stock, -20~80^oC)

For tyrosine-phosphorylated proteins, add:

2mM Sodium orthovanadate and 10mM Na pyrophosphate

3. StaphA-Wash Buffer: (for beads washing)

StaphA containing 0.1% BSA (fraction V). Prepare fresh.

1. Wash beads twice with 10ml wash buffer (without inhibitors) and resuspend in wash buffer as 50% slurry. (for 500ug protein input, use 20ul slurry (10ug agarose beads).

(if supplied as powder, Swell proteinA sepharose in wash buffer 1hr on ice). Wash beads twice with 10ml wash buffer (without inhibitors) and resuspend at 20mg/80ul in wash buffer.)

2. Lyse 10cm plate with 0.5ml lysis buffer on ice after two washes with chilled PBS (for PY-proteins: 2mM NaVO3) by scraping. Vortex, spin 15min in centrifuge in order to clear lysate. Read protein concentration on samples diluted 1:10 in water. use 0.5-5mg protein for detection with phosphotyrosine specific antibodies.
3. Adjust 500ug protein equivalent lysate volume with Lysis buffer to 1ml. Add 2ug(10ul) anti-ErbB3 (Santa Cruz sc285, rabbit polyclonal, 0.2mg/ml). Mix, incubate on ice or 4^oC for 1hr (rotation is not necessary at this step).
4. Add 20ul 50% Protein G-Agarose beads slurry and rotate at 4^oC for 1 hr.
5. Wash immunoprecipitates 4 times with StaphA buffer: spin at 4^oC at highest speed (14,000rpm) for 1-2min. aspirate supernatant until 2mm above beads pellets. add 1ml buffer and vortex (vigorously) and repeat.
6. To 10ul beads pellets, add 18ul H2O and 7ul 5XSB to get 1X. Vortex and heat at 95^oC for 5min, cool to RT, briefly spin, vortex, spin (14,000rpm 5min). Load 20ul (for phosphotyrosine) and 5ul (for ErbB3 loading control) onto 7.5% PAGE gel and proceed transfer and Western blot.