RIPA Lysis Buffer (Strong) 

MCE RIPA (Radio-Immunoprecipitation Assay) Lysis Buffer (Strong) is a widely used lysis and wash buffer for reporter assays, protein kinase assays, immunoassays and protein purification.

RIPA Lysis Buffer (Strong) consists of 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and general protease and phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride, EDTA). If desired, add MCE Protease Inhibitor Cocktail (HY-K0010) and MCE Phosphatase Inhibitor Cocktails (e.g., HY-K0021, HY-K0022, and HY-K0023).

Stored at -20°C, and is stable for up to 12 months.

1. Procedure for Cultured Cells

1.1 Pipette proper volume of RIPA Lysis Buffer and mix thoroughly. Add Protease Inhibitor Cocktail to lysis buffer to prevent proteolysis. Add Phosphatase Inhibitor Cocktails to maintain phosphorylation status of proteins as needed. Incubate on ice for subsequent use.

1.2 For adherent cells: Carefully remove culture medium from adherent cells. Wash cells twice with cold wash solution, such as PBS, normal saline or serum-free culture medium, to remove residual medium. Add proper volume of cold RIPA Lysis Buffer, then stroke with pipette until the buffer immerses cells completely. Incubate on ice and shake slightly for 5-10 minutes.

For suspension cultured cells: Collect cells into a centrifuge tube. Centrifuge samples at 500 g for 5 minutes and discard the supernatants. Wash cells twice with cold wash solution, such as PBS, normal saline or serum-free culture medium, to remove residual medium. Add proper volume of cold RIPA Lysis Buffer, then stroke with pipette until the buffer immerses cells completely. Incubate on ice and shake slightly for 5-10 minutes.

Note: Generally, use 1 mL of RIPA Lysis Buffer for 0.5-5 × 10⁶ cells. The volume of added RIPA Lysis Buffercan be adjusted proportionally.

1.3 After lysis, centrifuge at 14,000 g for 5 minutes at 4°C. Transfer the supernatants to a new tube for further analysis. Aliquot and freeze in liquid nitrogen and stored at -80°C for future use. Avoid multiple freeze/thaw cycles.

2. Procedure for Tissue Lysis

2.1 Pipette proper volume of RIPA Lysis Buffer and mix thoroughly. Add Protease Inhibitor Cocktail to lysis buffer to prevent proteolysis. Add Phosphatase Inhibitor Cocktails to maintain phosphorylation status of proteins as needed. Incubate on ice for subsequent use.

2.2 Cut tissue sample into small pieces on ice quickly to minimize protein degradation.

2.3 Add 150-250 μL cold RIPA Lysis Buffer per 20 mg of tissue sample and homogenize using electric homogenizer. Add more Lysis buffer if tissue is not completely lysed.

2.4 Transfer complete homogenized sample into a centrifuge tube. Incubate on ice and shake slightly for 5-10 minutes.

2.5 After lysis, centrifuge at 14,000 g for 5 minutes at 4°C. Transfer the supernatants to a new tube for further analysis. Aliquot and freeze in liquid nitrogen and stored at -80°C for future use. Avoid multiple freeze/thaw cycles.

Note: 20 mg of frozen mouse liver tissues may yield 15-25 mg/mL protein.