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Cell Lysate Preparation - NETN Buffer Method

This protocol outlines cell lysate preparation using the NETN method. This includes the recipes for reagents and the procedures for adherent cells and suspension culture.

Bethyl now sells NETN buffer. The buffer is manufactured to duplicate buffers used daily at Bethyl Laboratories while performing the high-quality antibody validation work that has become associated with our company.

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Materials:

  • Mammalian cells grown in adherent (100 mm dish) or suspension culture
  • Ice cold NETN Lysis Buffer
  • Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (100X) (Thermo Scientific #78442)
  • Ice cold PBS
  • Ice

Recipes:


NETN Lysis Buffer (store at 4 °C up to 1 month)

Stock Volume [Final]
5 M NaCl 5 mL 250 mM
0.5 M EDTA, pH 8.0 1 mL 5 mM
1 M Tris-HCl, pH 8.0 5 mL 50 mM
NP-40 (IGEPAL CA-630) 0.5 mL 0.5%
dH2O 88.5 mL  

NETN Lysis Buffer with Inhibitors (make fresh and keep on ice)

Stock Volume [Final]
Ice cold NETN Lysis Buffer 10 mL  
100X Halt Protease Phosphatase Inhibitor Cocktail 0.1 mL 1X

PBS (store at 4 °C up to 1 month)

Ingredients   [Final]
NaCl 8.0 g 137 mM
KCl 0.20 g 2.7 mM
NaH2PO4 0.23 g 1.9 mM
Na2HPO4 0.12 g 0.8 mM
dH2O 1 L  

Procedures:

Adherent cells

  1. Culture adherent cells to approximately 80% confluence on 100mm polystyrene tissue culture plates. Cells should be in log phase growth and healthy.
  2. Aspirate or decant media and keep plates on ice for all steps.
  3. Wash cell monolayer gently one time with 10 ml ice cold PBS. Aspirate excess PBS.
  4. Add 200 to 400 µl of NETN Lysis Buffer with Inhibitors to each plate and swirl to distribute buffer. If harvesting multiple plates of the same cell type, 0.5 to 1 ml of Lysis Buffer can be used to sequentially lyse at least 5 to 7 plates; this results in a higher concentration of protein in the final lysate. The optimal volume of lysis buffer should be empirically determined for each cell type to ensure efficient lysis as well as an optimal final concentration of protein in the lysate.
  5. Using a cell scraper or silicone spatula, scrape the cells and transfer the lysate to a 15 ml conical using a 1 ml pipette and tip.
  6. Incubate the lysate on ice for 30 minutes.
  7. Centrifuge at 13,000 x g for 5 minutes at 4 °C.
  8. Collect the supernatant (avoiding the pellet) into new microtubes.
  9. Determine protein concentration by the bicinchoninic acid method (Pierce 23228).
  10. Aliquot and store lysate at -20 °C avoiding multiple freeze/thaw cycles.

Suspension culture

  1. Culture cells to a density of 1-2 million cells/ml. Cells should be in log phase growth and healthy.
  2. Pellet cells in a conical tube by spinning at 300 x g for 5 minutes at room temperature.
  3. Aspirate or decant media; keep cells on ice for all steps.
  4. Wash pellet one time with 10 ml ice cold PBS.
  5. Spin 300 x g for 5 minutes. Decant the PBS wash and aspirate the excess supernatant.
  6. Add 10 to 100 µl of NETN Lysis Buffer with Inhibitors per 2 x 106 cells. The optimal volume of lysis buffer should be empirically determined for each cell type to ensure efficient lysis as well as an optimal final concentration of protein in the lysate.
  7. Incubate the lysate on ice for 30 minutes.
  8. Centrifuge at 13,000 x g for 5 minutes at 4 °C.
  9. Collect the supernatant (avoiding the pellet) into new microtubes.
  10. Determine protein concentration by the bicinchoninic acid method (Pierce 23228).
  11. Aliquot and store lysate at -20 °C. Avoid multiple freeze/thaw cycles.

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