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Protocols

RIPA Lysis

 

Materials:

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

 

Recipes:

  • RIPA Lysis Buffer (store at 4°C up to 1 month)
Stock Volume [Final]
5 M NaCl 3 mL 150 mM
0.5 M EDTA, pH 8.0 1 mL 5 mM
1 M Tris, pH 8.0 5 mL 50 mM
NP-40 (IGEPAL CA-630) 1 mL 1.0%
10% sodium deoxycholate 5 mL 0.5%
10% SDS 1 mL 0.1%
dH2O 84 mL  

 

  • RIPA Lysis Buffer with Inhibitors (make fresh and keep on ice)
Stock Volume [Final]
Ice cold RIPA Lysis Buffer 10 mL  
100X Halt Protease Phosphatase Inhibitor CockTail 0.1 mL 1X

 

  • PBS (store at 4°C up to 1 month)
Ingredients Volume [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 500 µl of RIPA 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 plates; this results in a higher concentration of protein in the final lysate. The amount 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.
  6. Incubate the lysate on ice for 15 minutes.
  7. Sonicate the lysate (Branson Digital Sonifier set at 50% amplitude) three times for two seconds each with at least one minute rest on ice between each two-second pulse. If lysate is still viscous repeat sonication.
  8. Incubate the lysate an additional 15 minutes.
  9. Centrifuge at 13,000 x g for 5 minutes at 4°C.
  10. Collect the supernatant (avoiding the pellet) into new microtubes.
  11. Determine protein concentration by the bicinchoninic acid method (Pierce 23228).
  12. Aliquot and store lysate at -20ºC 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 5 to 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 RIPA Lysis Buffer with Inhibitors per 1 x 106 cells. The amount 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 15 minutes.
  8. Sonicate the lysate (Branson Digital Sonifier set at 50% amplitude) three times for two seconds each with at least one minute rest on ice between each two-second pulse. If lysate is still viscous repeat sonication.
  9. Incubate the lysate an additional 15 minutes.
  10. Centrifuge at 13,000 x g for 5 minutes at 4ºC.
  11. Collect the supernatant (avoiding the pellet) into new microtubes.
  12. Determine protein concentration by the bicinchoninic acid method (Pierce 23228).
  13. Aliquot and store lysate at -20ºC. Avoid multiple freeze/thaw cycles.