This document provides a protocol for preparing radioimmunoprecipitation assay (RIPA) cell lysate from adherent cells. It describes making a RIPA lysis buffer containing Tris-HCl, EDTA, EGTA, Triton X-100, sodium deoxycholate, SDS and NaCl. Cells are washed, scraped into the buffer, lysed, and centrifuged to isolate proteins in the supernatant. The supernatant can be stored at -20°C or used immediately for applications like protein assays and purification. Following the protocol yields a protein lysate from adherent cells for use in analytical procedures.
This document provides a protocol for preparing radioimmunoprecipitation assay (RIPA) cell lysate from adherent cells. It describes making a RIPA lysis buffer containing Tris-HCl, EDTA, EGTA, Triton X-100, sodium deoxycholate, SDS and NaCl. Cells are washed, scraped into the buffer, lysed, and centrifuged to isolate proteins in the supernatant. The supernatant can be stored at -20°C or used immediately for applications like protein assays and purification. Following the protocol yields a protein lysate from adherent cells for use in analytical procedures.
This document provides a protocol for preparing radioimmunoprecipitation assay (RIPA) cell lysate from adherent cells. It describes making a RIPA lysis buffer containing Tris-HCl, EDTA, EGTA, Triton X-100, sodium deoxycholate, SDS and NaCl. Cells are washed, scraped into the buffer, lysed, and centrifuged to isolate proteins in the supernatant. The supernatant can be stored at -20°C or used immediately for applications like protein assays and purification. Following the protocol yields a protein lysate from adherent cells for use in analytical procedures.
This document provides a protocol for preparing radioimmunoprecipitation assay (RIPA) cell lysate from adherent cells. It describes making a RIPA lysis buffer containing Tris-HCl, EDTA, EGTA, Triton X-100, sodium deoxycholate, SDS and NaCl. Cells are washed, scraped into the buffer, lysed, and centrifuged to isolate proteins in the supernatant. The supernatant can be stored at -20°C or used immediately for applications like protein assays and purification. Following the protocol yields a protein lysate from adherent cells for use in analytical procedures.
Radio Immunoprecipitation Assay (RIPA) Cell Lysate Preparation
Introduction Radioimmunoprecipitation Assay Buffer (RIPA) is used to lyse cells and tissues for use in radioimmunoprecipitation, protein assays, protein purification and other analytical procedures. Due to its ionic detergent composition, RIPA can disrupt nuclear membranes and solubilize cytoplasmic proteins, resulting in a high protein yield. This protocol outlines the preparation of RIPA buffer and its use with adherent cells, resulting in a protein lysate that can be used immediately or can be stored for future use.
Method 1. Prepare the RIPA Lysis Buffer. Add 1mM PSMF immediately before use.
2. Remove all media from the tissue culture dish.
3. Wash cells twice with PBS gently, pouring off excess into waste beaker.
4. Carefully soak up any extra PBS with an appropriate lab wipe.
5. Add 500 µl of RIPA Lysis Buffer to the culture dish.
6. Use a cell scraper to scrape cells from the bottom of the dish.
7. Pass cell lysate through pipette 20 times to form a homogeneous lysate.
8. Transfer lysate to 1.5 ml microcentrifuge tube.
9. Allow samples to stand for 5 minutes at 4°C.
10. Centrifuge the resulting mixture at 14,000 g for 15 minutes at 4°C to separate cell debris from protein.
11. Transfer supernatant to a new tube and store at -20°C.
Note: To ensure a protease-free supernatant if you perform protein purification, add one of our ProBlock GoldTM Protease Inhibitor Cocktail to your suspension, available in multiple formats for any application.
References Janes, K. A. (2015). An analysis of critical factors for quantitative immunoblotting. Science Signaling, 8(371). Doi:10.1126/scisignal.2005966.
Monroy, F. (n.d.). RIPA Buffer Protocol. Retrieved April 26, 2018, from http://www2.nau.edu/fpm/documents/RIPAbuffer.pdf. Northern Arizona University.
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