Oct 30, 2017

Public workspaceTandem affinity purification of a bait host protein in presence of a viral protein V.1

PLOS Pathogens
DOI
dx.doi.org/10.17504/protocols.io.jeqcjdw
  • Benoit Besson1,
  • Florian Sonthonnax1,
  • Magalie Duchateau1,
  • Youcef Ben Khalifa1,
  • Florence Larrous1,
  • Hyeju Eun2,
  • Véronique Hourdel1,
  • Mariette Matondo1,
  • Julia Chamot-Rooke1,
  • Regis Grailhe2,
  • Hervé Bourhy1
  • 1Institut Pasteur;
  • 2Institut Pasteur Korea
Benoit Besson
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DOI: dx.doi.org/10.17504/protocols.io.jeqcjdw
External link: https://doi.org/10.1371/journal.ppat.1006697
Protocol Citation: Benoit Besson, Florian Sonthonnax, Magalie Duchateau, Youcef Ben Khalifa, Florence Larrous, Hyeju Eun, Véronique Hourdel, Mariette Matondo, Julia Chamot-Rooke, Regis Grailhe, Hervé Bourhy 2017. Tandem affinity purification of a bait host protein in presence of a viral protein. protocols.io https://dx.doi.org/10.17504/protocols.io.jeqcjdw
Manuscript citation:
Besson B, Sonthonnax F, Duchateau M, Khalifa YB, Larrous F, Eun H, Hourdel V, Matondo M, Chamot-Rooke J, Grailhe R, Bourhy H (2017) Regulation of NF-κB by the p105-ABIN2-TPL2 complex and RelAp43 during rabies virus infection. PLoS Pathog 13(10): e1006697. doi: 10.1371/journal.ppat.1006697
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
Created: August 15, 2017
Last Modified: March 09, 2018
Protocol Integer ID: 7344
Abstract
1st purification: a-FLAG (A2220; Sigma-Aldrich)
2nd purification: GFP TRAP_A (ChromoTek)
Day 1 - plate cells
Day 1 - plate cells
  1. Plate 5. 106 (± 1/6,5 of a confluent T150) HeLa cells in 20 mL of DMEM 10% FBS, on 75cm flask. 10 flasks/condition.
  2. Incubate at 37oC, 5% CO
Day 2 - transfection (per flask)
Day 2 - transfection (per flask)
  1. Lipofectamine 2000 mix: 60 ul of Lipo + 940 ul of DMEM.
  2. DNA Mix: 10 ug of DNA (3,34 µg for V5-tagged viral protein + 6,66 µg for FLAG-GFP tagged bait) in 1mL of DMEM (final volume).
  3. Add 1 to 2, mix.
  4. Incubate 30 min at room temperature.
  5. Remove 5 mL of DMEM from the flask and add the 2 mL of transfection (final volume of 17 mL)
  6. Incubate 24 hours at 37oC, 5% CO
Day 3 - buffer preparation
Day 3 - buffer preparation
50 mL IP buffer (1 week, 4°C)
45 mL of H2O
3mL from NaCl 5M stock (300 mM)
1,5 mL from Tris-HCl pH 7,4, 1M stock (30 mM)
0,5 g of Triton (1 %)
1 mM EDTA (supplemented with the tablet of protease inhibitor)
* Add 1 tablet of protease inhibitor Complete (Roche) / 50 mL lysis buffer.
* Add 1 tablet PhosStop (Roche) / 10 mL lysis buffer.
Day 3 - harvesting cells
Day 3 - harvesting cells
  1. Remove culture medium.
  2. Wash 2X with 1X PBS.
  3. Add 2 mL of PBS per flask.
  4. Scrap cells with cell scraper.
  5. Collect all in one 50 mL tube.
  6. Spin 3000 g, 10 min, 4°C
  7. Remove supernatant.
  8. Add 5 mL of IP buffer.
  9. Incubate 30 min, 4oC, under gentle rotation.
  10. Transfer in 2 mL tubes.
  11. Spin 13,000 g, 30 min, 4oC.
  12. Transfer supernatants to 15 mL tube 
  13. Determine protein concentration with a BCA kit. Follow instructions from provider.
Day 3 - Anti-FLAG IP
Day 3 - Anti-FLAG IP
  1. Incubate 30 mg protein with 200 µL agarose A/G beads (50% beads) in 15 mL tube.
  2. Incubate 1 hour, 4oC, under gentle rotation.
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Prepare anti-FLAG
  1. Wash 200 µL beads ANTI-FLAG M2 beads (A2220; Sigma-Aldrich) with 1 mL IP buffer in 1,5 mL tubes.
  2. Spin 1 minute, 5000 g, 4o
  3. Repeat 2X.
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  1. Spin 2500 g, 5 min, 4o
  2. Transfer supernatant to 200 µL ANTI-FLAG M2 beads in 15 mL tube.
  3. Incubate overnight, 4oC, under gentle rotation.
Day 4 - buffer preparation
Day 4 - buffer preparation
100 mL TBS
92 mL of H2O
5 mL from Tris-HCl, pH 7.4, 1M stock (50 mM)
3 mL from NaCl 5M stock (150 mM)
TBS-ETDA
50 / 10 mL TBS
20 / 4 µL from EDTA 0,5 M (0,2 mM)
10 mL TBS-inhibitors
TBS
* Add 1 tablet of protease inhibitor Complete EDTA-free (Roche) / 10 mL TBS.
* Add 1 tablet PhosStop (Roche) / 10 mL TBS.
Prepare 3X Flag peptide
Dissolve 3X flag peptide (F 4799; Sigma-Aldrich) in TBS-inhibitors at concentration 5 mg/mL.
Aliquot and store at -20oC. Repeated freezing and thawing is not recommended.
Day 4 - FLAG elution
Day 4 - FLAG elution
  1. Spin 5000 g, 4oC, 1 min.
  2. Discard supernatant.
  3. Wash 3X with 1000 µL IP buffer (spin 1 min at 5000 g, 4°C).
  4. For elution, add 100 µL of 3X flag elution solution (1 mg/mL) to each sample (dilution with 10 mL TBS-inhibitors).
  5. Rotate 1 hour, 4o
Day 4 - Anti-
Day 4 - Anti-
Prepare GFP TRAP
  1. Wash 100 µL beads with 1 mL TBS-EDTA.
  2. Spin 5 minutes, 2500 g, 4oC.
  3. Repeat 3X.
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  1. Centrifuge resin for 5 minutes at 2500 g, 4o
  2. Transfer supernatant to fresh tubes. E1, E2, E3
  3. Repeat steps 8 to 11, 3X.
  4. Transfer E1-3 with 100 µL GFP TRAP_A beads (ChromoTek).
  5. Top up to 1 mL with TBS-EDTA.
  6. Rotate 1 hour, 4o
  7. Centrifuge resin for 2 minutes at 2500 g, 4o
  8. Remove supernatant.
  9. Wash 3X with 1000 µL TBS-EDTA.
  10. Re-suspend beads in 100 µL 2X LDS (NUPAGE, 4X), 1X Reducing agent (NUPAGE, 10X). 95°C, 10 min.
  11. 12500 g, 2 min.
  12. Freeze protein elution at -20o
  13. Use 20 µL to run a western blot and check the presence/absence of controls.
  14. Run a keratin-free SDS-PAGE with 2 x 40 µL.
  15. Proceed to mass spectrometry.