Grow up 5ml LB with Amp o/n.
Add to 45ml LB with Amp
37o shake 2.5 ? 3 hrs, till OD600 0.4-0.8
Put bottles in room temperature water for 10 min to cool down.
Add 100ul 0.2M IPTG to 0.4mM final
Shake 30o 2hr
Pellet bacteria, decant sup, invert to drain
Resuspend in 1ml NETN 0.2mM PMSF / 50ml LB
PMSF, stock 10mM
NETN: 20mM Tris-Cl (pH8)
100mM NaCl
1mM EDTA
0.5% NP40
store at 4o
Vortex to mix well
Sonicate at scale 5 for 15sec. Keep on ice. For 10ml Corning tubes, use scale 7
Spin 4o, 5min
Transfer supernatant to a new tube.
To each lysate, add 60ul 50% Glutathione-Sepharose 4B
Pepette 400 ul Sepharose stock (75%)
Spin 1000rpm 5min, discard supernantant
Wash 3x300ul NETN
Resuspend in 300ul NETN to get 50% beads
Mix in cold room for 2 hours, slowly whirl
Pellet beads by brief centrifugation, carefully discard supernatant
Wash 3x1ml NETN/PMSF
Wash 2x1ml Elution Buffer (50mM Hepes, pH7.9, 40mM KCl, 1mM EDTA 1mM DTT)
Elute proteins by mix beads with 60ul each
Elution buffer
5mM Glutathione, (for 10mM, use 3.07mg/ml)
1mM DTT
Slowly swirl at RT 1hr
Quick spin to pellet, transfer supernatant to a new tube
Re-elute with 60ul each
NETN
5mM Glutathione
1mM DTT
Slowly swirl at RT 30min
Quick spin, combine supernatant, spin and transfer supernatant twice to avoid any residual beads. total is 120ul now.
Dialyze vs 50% glycerol/10mM Hepes, pH7.5/ 40mM KCl/ 1mM EDTA/ 1mM DTT/ 1mM PMSF in cold room for 2hr or o/n, store at ?20o
Proteins can also be concentrated in a Centriprep-30 concentrator. The pore size of the membrane in the Centriprep-30 allows glutathione to pass into the aqueous compartment. PBS can be added to the protein concentrate and the concentration procedure can be repeated.
Thinking aliquot and save at ?80o
Run 12% SDS-PAGE
