Mar 20, 2024

Public workspaceRecombinant αS purification Protocol

DOI
dx.doi.org/10.17504/protocols.io.q26g7popkgwz/v1
  • 1ASAP - Team Lee
Jane Balster
Open access
DOI: 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
Abstract
This protocol details the recombinant αS purification.
Attachments
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
Culture Growth and Induction
1d 14h 5m
Day 1: Inoculate Amount5 mL of LB/Amp [Amount100 undetermined : Amount1 µL of Amount100 undetermined Amp stock (freezer) for every Amount1 mL of culture] with 1 colony from LB/Amp plate and put in a shaker at Temperature37 °C w/Shaker250 rpm DurationOvernight .

  • Modification:

  1. Inoculate Amount10 mL of LB/Amp with 1 colony (allows more rapid growth).

  • If no viable plate:

  1. Take a stab from glycerol stock (Temperature-80 °C Freezer) and grow DurationOvernight .
  2. Streak LB/Amp agar plate with DurationOvernight culture.
8h
Centrifigation
Overnight
Temperature
Day 2: Inoculate Amount500 mL of LB/Amp (Amount100 undetermined : Amount1 µL of Amount100 undetermined stock for every Amount1 mL of culture) with Amount5 mL of DurationOvernight culture and put in shaker at Temperature37 °C until O.D = 0.5 – 0.6 (~1.5h -Duration03:00:00 )

  • Modification:

  1. Inoculate Amount1000 mL of LB/Amp with Amount10 mL of DurationOvernight culture and put in shaker.
  2. Nano-drop:

  • Use Amount2 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
Centrifigation
Pipetting
Overnight
Induce with IPTG (Amount400 undetermined : Amount6.4 µL of Concentration0.5 Molarity (M) ) for every Amount10 mL of culture) for 4h-Duration06:00:00 at Temperature37 °C .
6h
Temperature
Spin down culture at Centrifigation4600 x g, 4°C, 00:20:00 in Amount500 mL buckets (max volume Amount350 mL ) in JLA-10.5 rotor (blue rotor, standing centrifuge).


Note
***First Stopping Point: Pellet can be frozen at Temperature-20 °C . For > Amount500 mL culture, split pellet into Amount500 mL equivalent fractions***

20m
Centrifigation
Temperature
Day 3: Resuspend in Amount50 mL of lysis buffer. Add 1 tablet of protease inhibitor cocktail (NOT MINI) and Amount50 µL of Amount500 undetermined PMSF (Amount500 undetermined stock in floor Temperature-20 °C ; Final conc. Amount500 undetermined ; incubate at Temperature37 °C without shaking for 35min-Duration00:40:00 .

Lysis Buffer:
AB
NaOH40 mM
Tris pH 8.020 mM
EDTA1 mM
Triton X-1000.1%
40m
Incubation
Pipetting
Temperature
Add Amount500 µL of Concentration1 Molarity (M) MgCl2, Amount500 µL of Concentration1 Molarity (M) CaCl2, and Amount20 µL of DNase from Roche (Amount200 µL total) and incubate at Temperature37 °C with Shaker250 rpm shaking forDuration01:00:00 hour.

1h
Incubation
Centrifigation
Pipetting
Add Amount1 mL Concentration0.25 Molarity (M) EDTA, mix well, and remove cellular debris by centrifugation at Centrifigation16900 x g, 00:15:00 (JA 25.50 rotor).
15m
Centrifigation
Pipetting
Mix
Add Amount0.116 g of ammonium sulfate per mL of supernatant and stir at Temperature4 °C for Duration01:00:00 . Centrifuge at Centrifigation20000 x g for Duration00:30:00 , use new tubes. (~Amount5.8 g ; Toss pellet).
1h 30m
Centrifigation
Mix
Temperature
Add Amount0.244 g of ammonium sulfate per mL of supernatant and stir at Temperature4 °C for Duration01:00:00 (or DurationOvernight ). Centrifuge atCentrifigation20000 x g for Duration00:30:00 , use new tubes. (~Amount12.9 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
Centrifigation
Overnight
Temperature
Day 4: Resolubilize in Amount25 mL s Buffer A and add PMSF to Amount1 undetermined . Stir in refrigerator for ~Duration00:30:00 .

Buffer A:
Thris pH 8.025 mM
NaCl20 mM
EDTA1 mM
30m
Mix
Dialyze against Amount1 L of Buffer A with Amount0.5 undetermined PMSF DurationOvernight .
8h
Overnight
Day 5: Filter through Amount0.22 undetermined 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 Temperature4 °C . SEE DETAILED QFF PROTOCOL

Buffer B (filtered):
AB
Tris pH 8.025 mM
NaCl1 M
EDTA1 mM
Temperature
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 Centrifigation4500 x g for ~Duration00:20:00 till final sample volume <= Amount1 mL (lower volume better e.g. Amount750 µL ).

Buffer C (filtered):
AB
Tris pH 825 mM
NaCl1 M
EDTA1 mM
20m
Centrifigation
Day 6: LPLC: Run over size exclusion column in Buffer C. aS will start to come off at ~Amount80 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 Amount5 undetermined ).
  • Freeze aliquots at Temperature-80 °C
Temperature