Mar 20, 2024

Recombinant αS purification Protocol

DOI
https://dx.doi.org/10.17504/protocols.io.q26g7popkgwz/v1
  • 1ASAP - Team Lee
  • Team Lee
Jane Balster
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g7popkgwz/v1
Protocol Citation: Jane Balster 2024. Recombinant αS purification Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7popkgwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 21, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95779
Keywords: ASAPCRN, recombinant αs purification protocol, recombinant αs purification, recombinant, protocol this protocol detail, protocol detail, protocol
Abstract
This protocol details the recombinant αS purification.
Attachments
Icon for Generation of recombinant aS and PFF protocol.docx
Generation of recomb...
22KB
Materials
Lysis Buffer:
AB
NaOH40 mM
Tris pH 8.020 mM
EDTA1 mM
Triton X-1000.10%
Buffer A:
Thris pH 8.025 mM
NaCl20 mM
EDTA1 mM
Buffer B (filtered) :
Tris pH 8.025 mM
NaCl1M
EDTA1mM
Buffer C (filtered) :
Tris pH 825mM
NaCl1M
EDTA1mM
Culture Growth and Induction
1d 14h 5m
Day 1: Inoculate 5 mL of LB/Amp [100 µL : 1 µL of 100 µL 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/250 rpm Overnight .

  • Modification:

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

  • If no viable plate:

  1. Take a stab from glycerol stock (-80 °C Freezer) and grow Overnight .
  2. Streak LB/Amp agar plate with Overnight culture.
8h
Day 2: Inoculate 500 mL of LB/Amp (100 µL : 1 µL of 100 µL stock for every 1 mL of culture) with 5 mL of Overnight culture and put in shaker at 37 °C until O.D = 0.5 – 0.6 (~1.5h -03:00:00 )

  • Modification:

  1. Inoculate 1000 mL of LB/Amp with 10 mL of Overnight culture and put in shaker.
  2. 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):




3h
Induce with IPTG (400 µL : 6.4 µL of 0.5 Mass Percent ) for every 10 mL of culture) for 4h-06:00:00 at 37 °C .
6h
Spin down culture at 4600 x g, 4°C, 00:20:00 in 500 mL buckets (max volume 350 mL ) in JLA-10.5 rotor (blue rotor, standing centrifuge).


Note
***First Stopping Point: Pellet can be frozen at -20 °C . For > 500 mL culture, split pellet into 500 mL equivalent fractions***

20m
Day 3: Resuspend in 50 mL of lysis buffer. Add 1 tablet of protease inhibitor cocktail (NOT MINI) and 50 µL of 500 µL PMSF (500 µL stock in floor -20 °C ; Final conc. 500 µL ; incubate at 37 °C without shaking for 35min-00:40:00 .

Lysis Buffer:
AB
NaOH40 mM
Tris pH 8.020 mM
EDTA1 mM
Triton X-1000.1%
40m
Add 500 µL of 1 Mass Percent MgCl2, 500 µL of 1 Mass Percent CaCl2, and 20 µL of DNase from Roche (200 µL total) and incubate at 37 °C with 250 rpm shaking for01:00:00 hour.

1h
Add 1 mL 0.25 Mass Percent EDTA, mix well, and remove cellular debris by centrifugation at 16900 x g, 00:15:00 (JA 25.50 rotor).
15m
Add 0.116 g of ammonium sulfate per mL of supernatant and stir at 4 °C for 01:00:00 . Centrifuge at 20000 x g for 00:30:00 , use new tubes. (~5.8 g ; Toss pellet).
1h 30m
Add 0.244 g of ammonium sulfate per mL of supernatant and stir at 4 °C for 01:00:00 (or Overnight ). Centrifuge at20000 x g for 00:30:00 , 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.
2h 30m
Day 4: Resolubilize in 25 mL s Buffer A and add PMSF to 1 µL . Stir in refrigerator for ~00:30:00 .

Buffer A:
Thris pH 8.025 mM
NaCl20 mM
EDTA1 mM
30m
Dialyze against 1 L of Buffer A with 0.5 µL PMSF Overnight .
8h
Day 5: Filter through 0.22 µL filter, Milex, Millipore.
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):
AB
Tris pH 8.025 mM
NaCl1 M
EDTA1 mM
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 ~00:20:00 till final sample volume <= 1 mL (lower volume better e.g. 750 µL ).

Buffer C (filtered):
AB
Tris pH 825 mM
NaCl1 M
EDTA1 mM
20m
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.
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 µL ).
  • Freeze aliquots at -80 °C