1) Grow 50ml of culture in LB or TB +antibiotic o/n at 37_C shaker.

2) Dilute culture in LB or TB +antibiotic 1:10

3) Grow 3hrs at 37_C.

4) Induce culture by adding 0.4 mM IPTG final concentration. (For 50 ml final culture add 20 μl of 1M IPTG).

5) Grow at 25_C for 1 hr.

6) Spin 5 min at 5 K

7) Wash pellet with half volume of cold H₂O. (For 50 ml culture use 25 ml H₂O.)

8) Wash bacteria again.

9) Resuspend in 1 ml of resuspension buffer per 50 ml of culture.

Resuspension Buffer- NETN + protease inhibitors

20 mM Tris pH 8.0

100 mM NaCl

1 mM EDTA

0.5% NP-40 or Triton-X

1 μg/ml Aproteinin

1 μg/ml PMSF

1 μg/ml Benzaminide

Note: Tim has 100x stock of protease inhibitors.

10) Sonicate for 2x for 10 seconds in cold room.

11) Pellet debris by spinning at 4_C (Easiest way is to put samples in multiple eppendorfs and spin in cold room at max for 2 min.

1) Pipette 400 μl of GST-Sepharose into Eppendorf

2) Spin beads 15 sec @ 8,000 RPM

3) Wash beads 2x with 4_C NETN

4) Resuspend pellet in 320 μl of NETN (Final volume will be ~550 μl) and put into 4 eppendorfs.

5) Add 300 μl of resuspension buffer and 75 μl of E. Coli. GST Lysate to each tube.

6) Incubate for 30 min while rocking in cold room.

7) Spin 15 seconds at 8,000 rpm.

8) Wash 3x with ~600 μl of resuspension buffer

9) Remove supernatant and resuspend in appropriate volume resuspension buffer. (You can add this directly to SDS load buffer for running on a gel.)