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5X RIPA Buffer with Protease Inhibitor Cocktail

MBS169028

20 mL

160 USD

Reagent

5X RIPA Buffer with Protease Inhibitor Cocktail

[5X RIPA Buffer with Protease Inhibitor Cocktail]

Upon receipt, aliquot and store Protease Inhibitor Cocktail at -20°C and avoid multiple freeze/thaw cycles. Store RIPA Buffer concentrate at room temperature.

Components: 1. RIPA Buffer (5X) (Part No. AKR-191): One bottle - 20 mL containing 125 mM Tris pH 7.6, 750 mM NaCl, 5% Igepal CA-630, 5% sodium deoxycholate, 0.5% SDS. 2. Protease Inhibitor Cocktail (100X) (Part No. 217205): One vial - 1 mL containing AEBSF, Aprotinin, Bestatin, E64, Leupeptin, and Pepstatin A in DMSO.

Preparation of the Reagents: 1X RIPA Buffer: Mix the 5X RIPA Buffer by inverting/vortexing the bottle a few minutes. Dilute the 5X RIPA Buffer to 1X with deionized water. Stir to homogeneity. Store at 4 degree C for up to 6 months. Protease Inhibitor Cocktail: Immediately before use dilute the Protease Inhibitor Cocktail 1:100 with 1X RIPA Buffer. Stir to homogeneity. Do not store diluted solutions containing protease inhibitors. Preparation of Samples: I. Adherent Cells . 1. Culture cells to approximately 80-90% confluence. 2. Aspirate the culture media and wash twice with cold PBS. 3. Add cold 1X RIPA Buffer to the cells, spreading evenly. Use 1 mL of buffer per 10-cm dish (56.7 cm2), maintaining on ice for 5 minutes. Cells may be scraped with a cell scraper. 4. Collect the lysate into a microcentrifuge tube. 5. Centrifuge for 10 minutes (14000 x g). 6. Transfer the supernatant to a clean tube and maintain at 4 degree C. Samples may be frozen at -80 degree C. II. Suspension Cells . 1. Collect the cells into an appropriate conical centrifuge tube. 2. Centrifuge for 5 minutes (600 x g). 3. Remove and discard the supernatant. 4. Wash the cells twice with cold PBS, centrifuging for 5 minutes between (600 x g). 5. Add cold 1X RIPA Buffer to the cell pellet. Use 1 mL of buffer per 40 mg of wet cell pellet. Gently resuspend the pellet by pipetting up and down. 6. Shake the suspension for 15 minutes at 4 degree C. 7. Centrifuge for 10 minutes (14000 x g). 8. Transfer the supernatant to a clean tube and maintain at 4 degree C. Samples may be frozen at -80 degree C.

Cell Signaling and Protein Biology; Protein Analysis Tools; Protein Isolation and Extraction; RIPA Buffer

RIPA Buffer continues to be a popular buffer to lyse plated and suspension cultured mammalian cells. The buffer extracts cytoplasmic, membrane, and nuclear proteins and is suitable for downstream assays such as reporter assays, protein assays, immunoassays and protein purification.

20 mL

160 USD