I. Harvesting cells from 6-well plate:

1. Rinse cells 2 times with cold PBS (2 ml/well).
2. Add to each well 350l triton X-100 cell lysis buffer containing PI’s and DTT. Lyse cells by incubating on ice for 15min with swirling every 2min. Scrape debris and pool like-samples in 1.5ml Eppie tube. Vortex for 15sec.
3. Spin lysates at 14,000rpm for 10min at 4C. (10,000g in eppendorf centrifuge).
4. Remove supernatant to fresh tube for pull-down and protein quantitation.
5. Perform BCA (Pierce) assay on 10µl of each pooled lysate.

II. Pull-down:

5. Add 40µl of A/G bead slurry (Santa Cruz) and 10µl of antibody per 0.1 to 2mg of protein lysate in a fresh tube. Bring volume to 1 ml with lysis buffer.
6. Incubate lysate/beads/antibody on rotating wheel for 90min at 4C.
7. Rinse 2x with 500µl triton X-100 lysis buffer by spinning the beads at 14,000rpm for 15sec, removing supernatant with 25G needle on a vacuum line, and resuspending them in TX buffer.
8. Rinse 2x with 500l digitonin lysis buffer (containing DTT and PI’s).
9. Remove all supernatant with a 25G needle and elute for 30min on ice using 60µl of digitonin lysis buffer (60-100 µl used for each 20 µl packed beads.)

10. Assay Kinase Activity at this point making sure beads are in solution when aliquots into the assay are made.

Reagents:
Triton lysis buffer (4oC):
Tris-Cl 20mM, pH7.4
NaCl 50mM
NaF 50mM
NaPPi 5mM
Sucrose 250mM
Triton X100 1% v/v

Just before use add:
DTT 1mM (1:1000 of 1M stock)

Protease inhibitor cocktail: 1mM PMSF
2µg/ml Leupeptin
25µg/ml soybean trypsin inhibitor
50µM Benzamidine

Digitonin lysis buffer (4oC):
Tris-Cl 20mM, pH7.4
NaCl 50mM
NaF 50mM
NaPPi 5mM
Sucrose 250mM

Just before use add:
DTT 1mM (1:1000 of 1M stock)
Protease inhibitor cocktail: 1mM PMSF