GST Pull Down

Reagents:

#1. IP/Lysis buffer (50 ml)

Volume Stock Final
1.5 ml 5M NaCl 150 mM
1 ml 1 M HEPES, pH7.2 20 mM
0.5 ml 1 M NaF 10 mM
40 ul 0.5 mM EDTA 0.4 mM
0.5 ml 100% triton X-100 1 %
0.5 ml 50 mM Na3VO4 0.5 mM
0.5 ml 100 mM PMSF 1 mM
0.5 ml 100 mM Aprotonin 1 mM
5 ul 5 mg/ml Leupeptin 0.5 ug/ml

#2. 100 mM PMSF: 0.085g in 5 ml isopropanol
Freshly dilute stock of PMSF 1:1000 into the volume of lysis buffer to be used for the initial incubation. A higher concentration of PMSF (1:100) specified in the original paper may be satisfactory but precipitates immediately upon addition to the solution.

Procedures

1) Spin cell extracts at 14K rpm in cold room for 10 min

2) Transfer supernatants to a 1.5 new microcentrifuge tube

3) Take Aproprate amount of cell extracts and mix with fresh cold IP/Lysis buffer to a total volume of 1 ml in a 1.5 new microcentrifuge tube

4) Add 15 ul of glutothine agrose beads carrying GST fussion proteins ( 1 Volume bead: 1 volume PBS)

5) Incubate at 4 C for 1-3 hour with continueous shaking

6) spin down beads in microcentrifuge for 5 seconds at 10K rpm

7) Suck off supernatant with vacume (BE CAREFUL, DO NOT take away the beads)

8) Wash the beads with 1 ml of cold IP/Lysis buffer for 3 time by repeating step 6 and 7.
9) Add 30 ul of 1 X SDS loading buffer.

10) Boil for 5 min before load on the SDS/page gel.