RIPA Lysis Buffer | Cell Lysis Experimentation | Proteins

Cell Lysate Preparation - RIPA Buffer Method

RIPA (Radioimmunoprecipitation Assay) - Buffer is a reagent used in cell lysis experimentation, to enable rapid, efficient solubilization of proteins. By minimizing non-specific protein binding, specific binding interactions can be easily studied and are commonly used in immunoprecipitation experiments.

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

Purchase RIPA Buffer from Bethyl Laboratories

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

Culture adherent cells to approximately 80% confluence on 100mm polystyrene tissue culture plates. Cells should be in log phase growth and healthy.
Aspirate or decant media and keep plates on ice for all steps.
Wash cell monolayer gently one time with 10 ml ice cold PBS. Aspirate excess PBS.
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.
Using a cell scraper or silicone spatula, scrape the cells and transfer the lysate to a 15 ml conical.
Incubate the lysate on ice for 15 minutes.
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.
Incubate the lysate an additional 15 minutes.
Centrifuge at 13,000 x g for 5 minutes at 4 °C.
Collect the supernatant (avoiding the pellet) into new microtubes.
Determine protein concentration by the bicinchoninic acid method (Pierce #23228).
Aliquot and store lysate at -20 °C avoiding multiple freeze/thaw cycles.

Suspension culture

Culture cells to a density of 1-2 million cells/ml. Cells should be in log phase growth and healthy.
Pellet cells in a conical tube by spinning at 300 x g for 5 minutes at room temperature.
Aspirate or decant media; keep cells on ice for all steps.
Wash pellet one time with 5 to 10 ml ice cold PBS.
Spin 300 x g for 5 minutes. Decant the PBS wash and aspirate the excess supernatant.
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.
Incubate the lysate on ice for 15 minutes.
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.
Incubate the lysate an additional 15 minutes.
Centrifuge at 13,000 x g for 5 minutes at 4 °C.
Collect the supernatant (avoiding the pellet) into new microtubes.
Determine protein concentration by the bicinchoninic acid method (Pierce #23228).
Aliquot and store lysate at -20 °C. Avoid multiple freeze/thaw cycles.