Team:Pitt/Protocols
Preparation of sensor extract
- Grow a small overnight culture of your desired cell line.
- This should ideally be done in SE media, but LB is fine.
- Ideal conditions: 37C, 225rpm, 5mL
- BL21 derivatives are the best cell lines, but DNAse/RNAse-deficient strains may be used as well.
- Inoculate a larger culture at OD 0.01 in SE media. This may contain an appropriate antibiotic. Grow at 37C, 180rpm until OD 0.8 – 1, then induce with .5 mM IPTG if appropriate. Grow an additional 3-4 hours.
- Growing longer at lower temperatures, such as 30C, is acceptable if the protein(s) expressed in the extract may be toxic.
- Note that cells can grow more densely in SE media than in LB, especially if greater aeration is provided (either with baffled flasks or higher rpm). Harvesting should occur slightly past mid-log phase.
- Pellet cells at 6,000g, 30min, 4C. Resuspend cells in cold SE RB (resuspension buffer) without pipetting (15 ml of RB per 1 gram of wet cells). Freeze in liquid nitrogen. Store at -80C up to 3 days.
- The cells can be washed with several spin/resuspension cycles with SE RB prior to freezing.
- Polypropylene tubes should be fine for freezing and storing.
- The cells should not be stored for longer than 3 days.
- Thaw cells on ice. Pellet at 5,000g, 20min, 4C. Resuspend in SE WB (5 ml of WB per 1 gram wet cells). Pellet again at 5,000g, 20min, 4C. Then resuspend carefully in SE WB (1 ml of WB per 1 gram wet cells).
- The first wash step can be skipped entirely, especially if additional wash steps were performed before freezing.
- The resuspension should be done without pipetting, but the resulting solution must be completely homogenous.
- Sonicate the concentrated cells on ice with 10 pulses of 10s each with 30s in between.
- The cells may become thick during this step. If this occurs, continue sonicating until the solution becomes less viscous.
- Centrifuge at 30,000g, 30m, 4C. Keep supernatant, discard dark green-black pellet. Centrifuge again at 30,000g, 30m, 4C. Again, keep the supernatant. The pellet will appear faint.
- The extract can be separated in microfuge tubes prior to this step, then recombined afterwards.
- If 30,000g is unattainable, 20,000g for 1 hour is sufficient as well.
- Add SE PB (0.1 mL for every 1 mL of supernatant). Incubate in the dark at 37C for 90m with rolling.
- Slow shaking is acceptable as well.
- Dialyze at 4C against SE DB with a 3.5-5kDa MWCO membrane (4 times against a 25x volume)
- 3 times against a 100x volume also works.
- Each dialysis step should be about an hour, with the final one being overnight.
- Centrifuge the extract at 4,000g for 10m. Aliquot the supernatant in appropriate volumes into microfuge tubes. Flash freeze, then store at -80C.
SE media protocol (1 liter)
- To 900mL of ddH2O, add the following:
- 5.6g KH2PO4
- 28.9g K2HPO4
- 10g yeast extract
- Autoclave the solution after dissolving the components.
- Meanwhile, add the following to 50mL of ddH2O:
- 15g dextrose (a.k.a D-glucose)
- 25mg thiamine hydrochloride (or 22.5mg thiamine)
- After completely dissolving the solids, add ddH2O to 60.0mL. Sterile filter the resulting solution. Add 40.0mL of the sterile filtered solution to the autoclaved media. Add ddH2O to 1.0L. Store at 4C.
SE DB protocol (1 liter)
- To 500mL of ddH2O, add the following:
- 10mL of 1M Tris-Ac buffer, pH 8.2 (stored at 4C)
- 3.00g of magnesium acetate tetrahydrate
- 5.89g of potassium acetate
- Add ddH2O to 1.0L. Autoclave, and store at 4C. If using for dialysis, can also be prepared fresh.
SE WB protocol (50 mL)
- Add 7.7mg of DTT to 50mL of autoclaved SE DB. Can be stored at 4C for up to a week.
SE RB protocol (50 mL)
- Add 27.3mg of βME to 50mL of SE WB. Can be stored at 4C, but better prepared fresh.
SE PB
Final concentrations of- 300 mM Tris Ac, pH 7.6
- 10 mM magnesium acetate
- 10 mM ATP
- 80 mM phosphoenol pyruvate (PEP)
- 5 mM DTT
- 40 µM Amino Acid mix
- 0.5% sodium azide
- 8 U/ml pyruvate kinase
The remainder of the protocol is identical to the European Molecular Biology Laboratory's protocol.