5X RIPA Buffer with Protease Inhibitor Cocktail - 20 ml

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    5X RIPA Buffer with Protease Inhibitor Cocktail - 20 ml

    CATALOG NUMBER: AKR-191

    QUANTITY: 20 mL

    COMPOSITION: 125 mM Tris pH 7.6, 750 mM NaCl, 5% Igepal CA-630, 5% sodium deoxycholate, 0.5% SDS.

    Storage

    Store RIPA Buffer concentrate at room temperature.

    Shelf Life

    1 year from date of receipt under proper storage conditions; aliquot to avoid multiple freeze thaw cycles

    Background

    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.

    Preparation of Reagents

    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 4ºC for up to 6 months.

    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 4ºC. Samples may be frozen at -80ºC.

    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 4ºC.

    7. Centrifuge for 10 minutes (14000 x g).

    8. Transfer the supernatant to a clean tube and maintain at 4ºC. Samples may be frozen at -80ºC.

    Recent Citations  

    1. Robinson, R. et al. (2022). Generation and characterization of a Müller-glial-cell-specific Il6ra knockout mouse to delineate the effects of IL-6 trans-signaling in the retina. Sci Rep. 12(1):17626. doi: 10.1038/s41598-022-22329-3.

    2. Zieren, R.C. et al. (2021). Defining candidate mRNA and protein EV biomarkers to discriminate ccRCC and pRCC from non-malignant renal cells in vitro. Med Oncol. 38(9):105. doi: 10.1007/s12032-021-01554-2.

    3. Grill, M. et al. (2021). Vocal Fold Fibroblasts in Reinke's Edema Show Alterations Involved in Extracellular Matrix Production, Cytokine Response and Cell Cycle Control. Biomedicines. 9(7):735. doi: 10.3390/biomedicines9070735.

    4. Dong, L. et al. (2020). Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium. J Extracell Vesicles. 10(2):e12044. doi: 10.1002/jev2.12044.

    5. Sharma, R. et al. (2020). Chronic alcohol exposure reduces acetylated histones in the sleep-wake regulatory brain regions to cause insomnia during withdrawal. Neuropharmacology. doi: 10.1016/j.neuropharm.2020.108332.

    6. Hortobagyi, D. et al. (2020). In vitro mechanical vibration down-regulates pro-inflammatory and profibrotic signaling in human vocal fold fibroblasts. PLoS One. 15(11):e0241901. doi: 10.1371/journal.pone.0241901.

    7. Sharma, R. et al. (2020). Short-term sleep deprivation immediately after contextual conditioning inhibits BDNF signaling and disrupts memory consolidation in predator odor trauma mice model of PSTD. Brain Res. doi: 10.1016/j.brainres.2020.147155.

    8. Teraoka, S. et al. (2020). Association of BRCA Mutantations and BRCAness Status With Anticancer Drug Sensitivities in Triple-Negative Breast Cancer Cell Lines. J Surg Res. 250:200-208. doi: 10.1016/j.jss.2019.12.040.

    9. Kirsch, A. et al. (2019). Development and validation of a novel phonomimetic bioreactor. PLoS One. 14(3):e0213788. doi: 10.1371/journal.pone.0213788.

    10. Li, G. et al. (2018). 4-1BB enhancement of CAR T function requires NF-κB and TRAFs. JCI Insight. 3(18). pii: 121322. doi: 10.1172/jci.insight.121322.

    Warranty

    These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions. THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CELL BIOLABS’s sole obligation and purchaser’s exclusive remedy for breach of this warranty shall be, at the option of CELL BIOLABS, to repair or replace the products. In no event shall CELL BIOLABS be liable for any proximate, incidental or consequential damages in connection with the products.  

    This product is for RESEARCH USE ONLY; not for use in diagnostic procedures.