10 October 2018

Reagents

Lysis Buffer (LysB) (pH to 7.5 then filter)

| Component | Concentration |
| --------- | ------------- |
| HEPES     | 25 mM         |
| NaCl      | 500 mM        |
| Imidazole | 20 mM         |
| Glycerol  | 5 %           |

Before use, add 2 mM βME.

Gel Filtration (GF) Buffer (pH to 7.5 then filter)

| Component | Amount |
| --------- | ------ |
| HEPES     | 25 mM  |
| NaCl      | 150 mM |
| Glycerol  | 5 %    |

Before use, add 0.5 mM TCEP.

Protocol

Grow and Harvest

  1. Grow 50 mL overnight in TB + antibiotic at 30 °C
    • inoculate either from plate or cell stock
  2. Inoculate 1 L of TB + antibiotic with 10 mL of overnight culture
    • If using carbenicillin, first spin down the overnight (3000g for 10 minutes), and resuspend in fresh TB. This helps remove secreted β-lactamase which will degrade the antibiotic.
  3. Grow at 37 °C until OD = 0.5, then turn temperature down to 18 °C
  4. After 30 minutes, add IPTG to 0.5 mM (0.5 mL of 1 M stock).
    • Before adding IPTG, take pre-induction sample (pellet the equivalent of 1 mL of culture at OD = 0.5, resuspend in 50µL sample buffer (SB))
  5. Induce overnight at 18 °C.
  6. Spin down culture in 1 L bottles at 3000g for 15 minutes
    • Take a post-induction samples
  7. Resuspend pellet in 20 mL of Lysis Buffer (LysB, don’t need βME) per liter of cells
  8. Add protease inhibitors
    • AEBSF/E-64/Leupeptin (200x)
    • Aprotinin (1000x)
    • Pepstatin (1000x)
    • Add lysozyme (50x, 12.5 mg/mL stock)
    • Add 1 µL of nuclease per 50 mL
  9. Store in -80 °C freezer

Purification (everything done at 4 °C)

  1. Thaw pellet in water batch, then transfer to metal beaker
  2. Sonicate on ice
    • 3 min total, 10 s on, 59 s off, 75% power
    • Lysate sample (1uL lysate + 19uL SB)
  3. Spin at 30,000 rcf for 30 minutes to pellet debris and unlysed cells.
  4. Carefully pour supernatant into beaker
    • Sup sample (1uL sup + 19uL SB)
  5. Remove superloop, rinse with H20, then fill with LysB (+ 2 mM βME) and attach back to FPLC
  6. Use 25 mL syringe to inject sample into loop (or load onto column using akta prime pump)
    • Collect flow through NiFT (1uL FT + 19uL SB)
  7. Load sample onto 5 mL HisTrap at flow = 1.5 mL/min
  8. Wash with 50 mL of LysB (add 2 mM βME first)
  9. Wash with 50 mL of LysB + 20 mM imidazole (40 mM final)
  10. Elute with LysB + 280 mM imidazole
    • NiEl sample (5 uL elution + 15 uL SB)
  11. Clean histrap
    • 15 mL 500 mM imidazole
    • 15 mL water
    • 5 mL stripping buffer (50 mM EDTA, 500 mM NaCl, 20 mM Tris 7.4)
    • Water
    • 1 M NaOH if necessary
    • 30% isopropanol if necessary
    • 15 mL water
    • 2.5 mL 0.1 M NiSO4
    • 15 mL water
    • 15 mL 20% EtOH
  12. Concentrate down to 0.5 mL in appropriate concentrator
  13. Equilibrate size exclusion column (SEC) with water, then GF (+ 0.5 mM TCEP)
  14. Attach and clean 1 mL loop
  15. Prepare fraction tubes
  16. Spin sample at max speed for 15 minutes (or filter through 0.2 µM spin filter)
  17. Load into loop
    1. Switch to inject
    2. Clean injection port with buffer
    3. Load syringe with sample, insert into port and push a tiny amount through
    4. Switch back to load, then push sample into loop
  18. Run program, watch for sample injection and fractionation
  19. Equilibrate column back into water then 20% EtOH
  20. Run gel of fractions
  21. Pool and concentrate
  22. Measure concentration (Bradford or A280)
  23. Aliquot and snap freeze in liquid nitrogen