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RIPA Lysis Protocol

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.

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152 views3 pages

RIPA Lysis Protocol

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.

Uploaded by

Alice Lesiv
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Protocol

TD-P Revision 3.0 Creation Date: 6/6/2016


Revision Date: 8/21/2018

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.

Materials
 Adherent cell culture
 Tris-HCl (GoldBio Catalog # T-095)
 EDTA (GoldBio Catalog # E-210)
 EGTA (GoldBio Catalog # E-217)
 Triton X-100
 Sodium Deoxycholate (GoldBio Catalog # D-070)
 SDS
 NaCl
 dH2O
 PMSF (GoldBio Catalog # P-470)
 PBS (GoldBio Catalog # P-271)
 ProBlock GoldTM Protease Inhibitor Cocktail (GoldBio Catalog # GB-108)

Preparation of RIPA lysis buffer:


 10mM Tris-HCl, pH 8.0
 1mM EDTA
 0.5mM EGTA
 1% Triton X-100
 0.1% Sodium Deoxycholate
 0.1% SDS
 140mM NaCl
 Dilute with dH2O
 This solution is stable at room temperature. Add 1mM PMSF immediately before use.

Gold Biotechnology
St. Louis, MO
Ph: (800) 248-7609
Web: www.goldbio.com
Email: [email protected]
Gold Biotechnology / FM-000008 TD-P Revision 3.0
RIPA Cell Lysate Preparation TD-P Date: 8/21/2018

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.

Associated Products
 Tris-HCl (GoldBio Catalog # T-095)
 EDTA (GoldBio Catalog # E-210)
 EGTA (GoldBio Catalog # E-217)
 Sodium Deoxycholate (GoldBio Catalog # D-070)
 PMSF (GoldBio Catalog # P-470)
 ProBlock GoldTM protease blocker (GoldBio Catalog # GB-108-2)
 PBS Tablets (GoldBio Catalog # P-271)

Gold Biotechnology
St. Louis, MO
Ph: (800) 248-7609
Web: www.goldbio.com 2
Email: [email protected]
Gold Biotechnology / FM-000008 TD-P Revision 3.0
RIPA Cell Lysate Preparation TD-P Date: 8/21/2018

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.

Gold Biotechnology
St. Louis, MO
Ph: (800) 248-7609
Web: www.goldbio.com 3
Email: [email protected]

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