Materials
| Bacterial cultures (pGEX/Ncd or motor of interest in Bl21(DE3)plysShost cells) |
| PB buffer = | 10 mM NaPO4 pH 7.2
1 mM EGTA
1 mM MgCl2
|
| HEM buffer = | 10 mM HEPES pH 7.2
1 mM MgCl2
1 mM EGTA
|
| PB buffer (or HEM buffer) + 50 mM NaCl |
| 200 mM PMSF (phenylmethylsulfonylflouride) in EtOH |
| Protease Inhibitor Cocktail (200X) = | 1 microgram/mL Pepstatin
1 microgram/mL Leupeptin
2 micrograms/mL Aprotinin
2 micrograms/mL TAME
|
Special Equipment
| Beckman TLA 100.3 rotor or comparable |
Procedure
| 1. |
Grow bacterial cultures containing pGEX/Ncd (or motor of interest) in BL21(DE3)plysShost cells at 37°C to OD550 = 0.8, then induce by adding 0.4 mM of IPTG followed by shaking at 22°C for 3.5 - 5 hours. |
| 2. |
Harvest cells. Wash in PB or HEM + 0.5 mM PMSF and centrifuge again. Flash freeze in liquid nitrogen and store at -80°C until use. |
| 3. |
Thaw cells and resuspend in PB (or HEM) + 50 mM NaCl + 1 mM PMSF + 1:200 protease inhibitor cocktail. Lyse by freezing in liquid nitrogen followed by thawing at 37°C until still cold. Transfer to ice. |
| 4. |
Digest bacterial DNA by adding 5 mM MgCl2, 1 mM DTT and 40 micrograms/mL DNAse I and incubate on ice for 15 - 20 min. Add more PMSF (to 2 mM) and protease inhibitor cocktail (to 1:100) midway through the incubation. Monitor DNA digestion by a reduction in viscosity of the lysate. |
| 5. |
Clarify the lysate by centrifugation at 27,000g(18K rpm) in a Sorvall SS-34 rotor for 20 min at 4°C followed by centrifugation at 155,000g(60K rpm) in a Beckman TLA 100.3 rotor for 20 min at 2°C. Withdraw the top half of the final supernatant for motility assays. |
S. Endow Laboratory