TITLE: Recombinant αS purification Protocol
AUTHORS: Jane Balster

[DESCRIPTION]
This protocol details the recombinant αS purification.

[STEPS]
SECTION: Culture Growth and Induction
1. Day 1: Inoculate 5 mL of LB/Amp [100 1527: 1 µL of 100 1524 Amp stock (freezer) for every 1 mL of culture] with 1 colony from LB/Amp plate and put in a shaker at 37 °C w/   .

Modification:

Inoculate 10 mL of LB/Amp with 1 colony (allows more rapid growth).

If no viable plate:

Take a stab from glycerol stock (-80 °C Freezer) and grow  .
Streak LB/Amp agar plate with   culture.
SECTION: Culture Growth and Induction
2. Day 2: Inoculate 500 mL of LB/Amp (100 1527: 1 µL of 100 1524 stock for every 1 mL of culture) with 5 mL of   culture and put in shaker at 37 °C until O.D = 0.5 – 0.6 (~1.5h -180 min)

Modification:

Inoculate 1000 mL of LB/Amp with 10 mL of   culture and put in shaker.
Nano-drop:

Use 2 µL drop.
Use LB or LB/Amp as a blank.

 3. Formula to determine the time it will take the culture to hit an OD of 0.5 (assuming 30 min doubling time):


 

SECTION: Culture Growth and Induction
3. Induce with IPTG (400 1527: 6.4 µL of 0.5 Molarity (M)) for every 10 mLof culture) for 4h-360 min at 37 °C.
SECTION: Culture Growth and Induction
13. LPLC: Run over Anion Exchange column HiTrap Q FF (program: SC IExAlphasS, Buffer A, filtered Buffer B), aS will start to come off at ~20% Buffer B). Keep samples at 4 °C. SEE DETAILED QFF PROTOCOL

Buffer B (filtered): 
 
 
SECTION: Culture Growth and Induction
4. Spin down culture at 4600 x g, 20 min, 4 °C in 500 mL buckets (max volume 350 mL) in JLA-10.5 rotor (blue rotor, standing centrifuge).


 

SECTION: Culture Growth and Induction
12. Day 5: Filter through 0.22 3190 filter, Milex, Millipore.
SECTION: Culture Growth and Induction
11. Dialyze against 1 L of Buffer A with 0.5 1536 PMSF  .
SECTION: Culture Growth and Induction
10. Day 4: Resolubilize in 25 mLs Buffer A and add PMSF to 1 1536. Stir in refrigerator for ~30 min.

Buffer A:
 
 
SECTION: Culture Growth and Induction
5. Day 3: Resuspend in 50 mLof lysis buffer. Add 1 tablet of protease inhibitor cocktail (NOT MINI) and 50 µL of 500 1536PMSF (500 1536 stock in floor -20 °C; Final conc. 500 1535; incubate at 37 °C without shaking for 35min-40 min.

Lysis Buffer: 
 
 
SECTION: Culture Growth and Induction
6. Add 500 µL of 1 Molarity (M) MgCl2, 500 µL of 1 Molarity (M) CaCl2, and 20 µL of DNase from Roche (200 µL total) and incubate at 37 °C with   shaking for60 minhour. 

SECTION: Culture Growth and Induction
9. Add 0.244 g of ammonium sulfate per mL of supernatant and stir at 4 °C for 60 min (or 60 min). Centrifuge at20000 x g for 30 min, use new tubes. (~12.9 g; **Keep pellet).

Quality Control Checkpoint: Pellet should be at bottom of tube. Excessive proteolysis is indicated by floating pellet or significant smearing of pellet along the side of the tube.
SECTION: Culture Growth and Induction
8. Add 0.116 g of ammonium sulfate per mL of supernatant and stir at 4 °C for 60 min. Centrifuge at 20000 x g for 30 min, use new tubes. (~5.8 g; Toss pellet).
SECTION: Culture Growth and Induction
7. Add 1 mL 0.25 Molarity (M)EDTA, mix well, and remove cellular debris by centrifugation at 16900 x g, 15 min (JA 25.50 rotor).
SECTION: Culture Growth and Induction
14. Concentrate αS positive fractions with Amicon Ultracel-3KD MWCO, Millipore, and exchange into filtered Buffer C. Perform during Day 6 equilibration.

Spin the samples at 4500 x g for ~20 min till final sample volume <= 1 mL (lower volume better e.g. 750 µL).

Buffer C (filtered):  
 
 
SECTION: Culture Growth and Induction
16. Concentrate aS positive fractions with Amicon Ultracel-3KD MWCO, Millipore, and exchange into sterile PBS.

BCA samples and aliquot at desired concentration/volume (recommended at 5 3189).
Freeze aliquots at -80 °C
SECTION: Culture Growth and Induction
15. Day 6: LPLC: Run over size exclusion column in Buffer C. aS will start to come off at ~80 mL. SEE DETAILED SEC PROTOCOL – Freeze tubes until Conc./BCA.