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Cultured cell lysis (Immunoblot & Immunoprecipitation)

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1. Background and purpose of the procedure

In this protocol, adherent cells are grown and treated in tissue culture flasks until ready to be used for protein sample preparation. To analyze proteins by immunoblotting, it is necessary to bring the proteins from the sample into soluble form using the buffers, salts, and detergents compatible with the gel electrophoresis procedure.

2. Materials and equipment

  • Adherent cells in cell culture flasks
  • Lysis Buffer 1X (see 3.b. Recipes)
  • Complete Protease Inhibitor (Roche, 1697498)
  • D-PBS, without Ca++ and Mg++ (Gibco, 14190144) 
  • Vacuum trap or aspiration
  • 1.5 mL or 2 mL microcentrifuge tubes
  • Cell scrapers (for adherent cells)
  • 1 mL syringe with 25-gauge needle
  • Microcentrifuge (refrigerated, 4°C)
  • Ice

3. Recipes

All stock solutions are prepared in ddH2O unless otherwise specified

2X Lysis Buffer

30 mM HEPES (Fisher BioReagents, BP310-1 – stock solution @ 1 M) [600 µL for 10 mL]
100 mM KCl (Fisher Chemical, AR Certified, P-4280-53 – stock solution @ 1 M) [2 mL for 10 mL]
20 mM NaCl (Fisher Chemical, AR Certified, S-3160-53 – stock solution @ 5 M) [80 µL for 10 mL]
2 mM MgCl2 [6▪H2O] (Fisher Chemical, M-0550-53 – stock solution @ 1 M) [40 µL for 10 mL]
5% Glycerol (Fisher BioReagents, Molecular Biology Grade, BP2294) [1 mL for 10 mL]
ddH2O qsp 10 mL [6.3 mL for 10 mL]

2X lysis buffer can be prepared in 100 mL batches and then aliquoted (5 or 10 mL in 15 mL centrifuge tubes) before being stored at -20°C for up to a year.

1X Lysis Buffer

2X lysis buffer [10mL for 20 mL]
0.5% n-Dodecyl-β-Maltoside (DDM) (Anatrace, D310-5 – stock solution @ 5%) [2 mL for 20 mL]
5 µM GDP (Sigma, G-7127 – stock solution @ 1 mM) [100 µL for 20 mL]
5 mM NaF (Fisher Chemical, AR Certified, S-3920-60 – stock solution @ 100 mM) [1 mL for 20 mL]
1 mM NaVO4 (Thermo Scientific Chemicals, 081104.22 – stock solution @ 1 M) [20 µL for 20 mL]
1X Complete Protease Inhibitor (Stock solution @ 25X [1 tablet + 2 mL ddH2O]) [800 µL for 20 mL]
ddH2O qsp 20 mL [6.1 mL for 20 mL]

1X lysis buffer can be stored at 4°C for up to 2 weeks.

Alternatively, NaF can be replaced with 1 µM Microcystine (Alexis, ALX-350-012-C500 – stock solution @ 1 mM) [20 µL for 20 mL]

4. Methods

  1. Discard the culture medium
  2. Wash cells twice with ice-cold D-PBS. Place the culture dishes/flasks on ice (all subsequent work has to done on ice or at 4°C). Ensure cell monolayer is drained from residual D-PBS
  3. Add pre-chilled 1X lysis buffer on adherent cells [100 to 200 µL per well (6-well plate) – 200 to 350 µL per T-25 flask, 400-600 µL per T-75 flask, or 1-1.5 mL per T-175 flask]
  4. Ensure complete spread of 1X lysis buffer over the cell monolayer in each well/flask
  5. Scrape cell monolayer with a cell scraper and transfer the lysate in a clean 1.5 or /2mL microcentrifuge tube for each condition
  6. Shear DNA mechanically by passing the homogenate multiple times through a 25-gauge needle mounted on a 1 mL syringe
  7. Incubate for 30 min at 4°C [vortex every 10 min]
  8. Centrifuge tubes at 13,000 rpm for 10 min at 4°C
  9. Transfer the supernatant in a clean 1.5 or /2mL microcentrifuge tube making sure not to disturb the pellet
  10. [Optional – Recommended for immunoprecipitation] Repeat the centrifugation of the collected supernatant at 13,000 rpm for 10 min at 4°C.
  11. Samples can be used fresh for dosage and immunoblot/immunoprecipitation or stored at -80°C for up to 6 months. For the analysis of protein complexes by immunoprecipitation, the use of a freshly prepared cell lysate is highly recommended.

5. References

Balabanian K, Brotin E, Biajoux V, Bouchet-Delbos L, Lainey E, Fenneteau O, Bonnet D, Fiette L, Emilie D, Bachelerie F. Proper desensitization of CXCR4 is required for lymphocyte development and peripheral compartmentalization in mice. Blood. 2012 Jun 14;119(24):5722-30. doi: 10.1182/blood-2012-01-403378. PMID: 22438253.

DeCaprio J, Kohl TO. Detergent Lysis of Tissue Culture Cells for Immunoprecipitation. Cold Spring Harb Protoc. 2017 Dec 1;2017(12):pdb.prot098558. doi: 10.1101/pdb.prot098558. PMID: 29196599.

6. Revision history

Revision #DatePrepared by
1.02023-07-25Cécile Pétigny, Elie Besserer-Offroy
Summary of modifications
Initial version of the protocol

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