RIPA Buffer is a lysis buffer that efficiently lyses cells and solubilizes proteins while minimizing protein degradation. It can be used to lyse both adherent and suspension cultured mammalian cells. The document provides detailed procedures for lysing both adherent and suspension cultured cells using RIPA Buffer. Key components of RIPA Buffer are also listed.
RIPA Buffer is a lysis buffer that efficiently lyses cells and solubilizes proteins while minimizing protein degradation. It can be used to lyse both adherent and suspension cultured mammalian cells. The document provides detailed procedures for lysing both adherent and suspension cultured cells using RIPA Buffer. Key components of RIPA Buffer are also listed.
RIPA Buffer is a lysis buffer that efficiently lyses cells and solubilizes proteins while minimizing protein degradation. It can be used to lyse both adherent and suspension cultured mammalian cells. The document provides detailed procedures for lysing both adherent and suspension cultured cells using RIPA Buffer. Key components of RIPA Buffer are also listed.
RIPA Buffer is a lysis buffer that efficiently lyses cells and solubilizes proteins while minimizing protein degradation. It can be used to lyse both adherent and suspension cultured mammalian cells. The document provides detailed procedures for lysing both adherent and suspension cultured cells using RIPA Buffer. Key components of RIPA Buffer are also listed.
Product Description • Cell scrapers for adherent cells (Product Code
Extraction of cellular proteins requires efficient cell lysis C 2802) and protein solubilization, while avoiding protein • Protease Inhibitor Cocktail for Mammalian Tissues degradation and/or interference with protein (Product Code P 8340) immunoreactivity and biological activity. • Phosphatase Inhibitor Cocktails • for Serine/Threonine Phosphatases and RIPA (Radio-Immunoprecipitation Assay) Buffer L-Isozymes of Alkaline Phosphatases (Product enables rapid, efficient cell lysis and solubilization of Code P 2850) proteins from both adherent and suspension cultured • for Tyrosine Protein Phosphatases, Acid and mammalian cells. It has long been a widely used lysis Alkaline Phosphatases (Product Code P 5726) and wash buffer for small-scale affinity pull-down applications, such as immunoprecipitation, since most Precautions and Disclaimer antibodies and protein antigens are not adversely RIPA Buffer is for laboratory use only, not for drug, affected by the components of this buffer.1 In addition, household, or other uses. Please consult the Material RIPA Buffer minimizes non-specific protein-binding Safety Data Sheet for information regarding hazards interactions to keep background low, while allowing and safe handling practices. most specific interactions to occur, enabling studies of relevant protein-protein interactions. Storage/Stability It is recommended to store the product at 2–8 °C. The RIPA Buffer is supplied as a ready to use solution that product is stable for 2 years when stored properly. requires no preparation. Protease and phosphatase inhibitors may be added to the lysis buffer as needed. Procedure One ml of the RIPA Buffer is sufficient to lyse cells from A. Procedure for lysis of adherent cells one 100 mm culture dish (0.5 to 5 x 107 cells) of most 1. Remove growth medium from the cells by adherent mammalian cell lines. decantation or aspiration. Component 2. Wash cells to remove residual medium. Slowly add RIPA Buffer (Product Code R 0278) – a volume of a physiological wash solution, such as 50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, DPBS, equal to the original medium volume being 1.0% Igepal CA-630 (NP-40), 0.5% sodium careful not to dislodge cells. Mix gently and deoxycholate, and 0.1% sodium dodecyl sulfate.1 remove the wash solution. Repeat the wash once in order to remove any other minor contaminants. Reagents and Equipment Required but not More washing steps can be done, but two is usually Provided sufficient to remove nearly all of the contaminants. (Product Codes are given where appropriate) • Centrifuge 3. After removal of the final wash solution from the • Appropriate centrifuge tubes cells, add an appropriate volume of RIPA Buffer • A physiological wash buffer (e.g., Dulbecco’s (1 ml for 0.5 to 5 x 107 cells). Incubate on ice or in Phosphate Buffered Saline [DPBS], Product Code a refrigerator (2–8 °C) for five minutes. D 8662) 4. Rapidly scrape the plate with a cell scraper to 4. After removal of the final wash solution from the remove and lyse residual cells. Transfer the cell cells, add an appropriate volume of RIPA Buffer lysate to a tube on ice. The lysate can either be (1 ml per 0.5 to 5 x 107 cells) to the cell pellet, and used immediately or quick-frozen in liquid nitrogen mix or vortex briefly to resuspend the cells and stored at –70 °C for future use. It is best to completely. Incubate on ice or in a refrigerator freeze the lysate before clarification, since the (2–8 °C) for five minutes. Vortex briefly to freeze-thaw cycle may cause some denatured resuspend and lyse residual cells. protein aggregates. 5. The lysate can either be used immediately or quick- 5. Clarify the lysate by centrifugation at 8,000 x g for frozen in liquid nitrogen and stored at –70 °C for 10 minutes at 4 °C to pellet the cell debris. future use.
Note: If a mucoid aggregate of denatured nucleic 6. Clarify the lysate by centrifugation at 8,000 x g for acids is present, carefully remove it with a 10 minutes at 4 °C to pellet the cell debris. micropipette before centrifugation. Note: If a mucoid aggregate of denatured nucleic 6. Carefully transfer the supernatant containing the acids is present, carefully remove it with a soluble protein to a tube on ice for micropipette before centrifugation. immunoprecipitation or other analysis. 7. Carefully transfer the supernatant containing the B. Procedure for lysis of suspension cultured cells soluble protein to a tube on ice for 1. Pellet the cells by centrifugation at 450 x g for immunoprecipitation or other analysis. 5 minutes. Reference 2. Carefully remove the medium from the cell pellet by 1. Antibodies a Laboratory Manual, Harlow, E. and decantation or aspiration. Lane, D., Cold Spring Harbor Press (Cold Spring Harbor, NY: 1988), p 449. 3. Wash the cells to remove residual medium. Add a volume of a physiological wash solution, such as ASC/NW/MAM 9/03 DPBS, equal to the original medium volume. Mix or vortex briefly to resuspend the cells completely. Centrifuge for 5 minutes at 450 x g to pellet the cells and carefully remove wash solution supernatant. Repeat the wash once in order to remove any other minor contaminants. More washing steps can be done, but two is usually sufficient to remove nearly all of the contaminants.
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