RIPA Lysis Buffer

Overview

Description

RIPA Lysis Buffer is a complete cell lysis solution reagent used for rapid and efficient total cell lysis and solubilization of proteins from both adherent and suspension cultured mammalian cells, effectively extracting cytoplasmic, nuclear and membrane proteins. Protein lysis can be finished in 60 minutes.

Background

RIPA lysis extraction buffer contains non-ionic and ionic detergents which are able to extract protein from wide variety of cell types and membrane structures. RIPA buffer ensures efficient cell lysis and protein solubilization preventing protein degradation and interference with protein immunoreactivity and biological activity. Since most antibodies and protein antigens are not adversely affected by the components of this solution, RIPA buffer-conducted protein extraction is compatible with various downstream immunoprecipitation and molecular pull-down assays, including reporter assays, protein assays, immunoassays and protein purification. RIPA buffer reagent minimizes non-specific protein-binding interactions to keep background low, while allowing most specific interactions to occur, enabling studies of relevant protein-protein interactions.

Important Product Information

• If desired, add protease inhibitor (Product No. AR1182) and phosphatase inhibitor (Product No. AR1183) to the lysis buffer to prevent proteolysis and maintain phosphorylation status of proteins.

• Some protein kinases and other enzymes may be sensitive to the components of the RIPA Lysis Buffer, resulting in their decreased activity. In such cases, prepare a RIPA Lysis Buffer that does not contain sodium deoxycholate and SDS.

Additional Materials Required

• Protease inhibitor (Product No. AR1182) and phosphatase inhibitor (Product No. AR1183)

• 2 ml microcentrifuge tubes

• Tissue homogenizer

• Microcentrifuge capable of spinning at 10,000 x g

• Cell scraper

Procedure for Lysis of Monolayer-cultured Adherent Mammalian Cells

Note: Pre-chill an appropriate volume of RIPA Lysis Buffer at 4°C. If desired, add protease inhibitor and phosphatase inhibitor to the lysis buffer immediately before use.

1. In a microcentrifuge tube, resuspend 5×10⁶ cells in the growth media by scraping the cells off the surface of the plate with a cell scraper. Centrifuge harvested cell suspension at 600xg for 5min, then carefully remove and discard the supernatant.

2. Resuspend the cells in chilled PBS. Centrifuge at 600xg for 5min, then carefully remove and discard the supernatant.

3. Add 0.5 mL of chilled RIPA lysis buffer to the cell pellet. Vortex briefly. Incubate on ice for 30 minutes.

4. Centrifuge samples at 14000xg for 10 minutes.

5. Transfer supernatant to a new tube for further analysis.

Note: RIPA lysis buffer can be added directly to the flask containing cells. Please see the following procedures.

1. Carefully remove culture medium from adherent cells.

2. Wash cells with chilled PBS. Carefully remove PBS.

3. Add chilled RIPA lysis buffer to the cells. Vortex briefly. Incubate on ice for 30 minutes. (For the volume of the lysis buffer, follow the instructions listed below)

4. Centrifuge samples at 14000xg for 10 minutes.

5. Transfer supernatant to a new tube for further analysis.

Procedure for Lysis of Suspension-cultured Mammalian Cells

Note: Pre-chill an appropriate volume of RIPA Lysis Buffer at 4°C. If desired, add protease inhibitor and phosphatase inhibitor to the lysis buffer immediately before use.

1. In a microcentrifuge tube, harvest 5×10⁶ cells by centrifugation at 600xg for 5min. Carefully remove and discard the supernatant.

2. Resuspend the cells in chilled PBS. Centrifuge at 600xg for 5min, then carefully remove and discard the supernatant.

3. Add 0.5 mL of chilled RIPA lysis buffer to the cell pellet. Vortex briefly. Incubate on ice for 30 minutes.

4. Centrifuge samples at 14000xg for 10 minutes.

5. Transfer supernatant to a new tube for further analysis.

Procedure for Lysis of Tissues

Note: Pre-chill an appropriate volume of RIPA Lysis Buffer at 4°C. If desired, add protease inhibitor and phosphatase inhibitor to the lysis buffer immediately before use.

1. Place the fresh tissue into chilled PBS and rinse several times. Mince the tissue into small pieces.

2. Add RIPA Lysis Buffer to the tissue at 10:1. (i.e., Add 10 mL chilled lysis buffer per gram of tissue.) Use a smaller volume of reagent if a more concentrated protein extract is required.

3. Homogenize for several minutes at high speed until no tissue chunks remain.

4. Incubate on ice for 30 minutes.

5. Centrifuge at ~10000 x g for 10 minutes.

6. Transfer supernatant to a new tube for further analysis.

Precautions

• All steps of protein lysis should be operated on ice or at 4°C.

• Use BCA Protein Assay kit (Product No. AR0146) to quantify lysed proteins. Bradford Protein Assay kit is not recommended.

• There might be some transparent gel complex containing genomic DNA in lysed proteins. The protein fractions can be used for further analysis after centrifugation if target proteins have little connection with genomic DNA. When detecting target proteins related closely to genomic DNA, sonicate gel complex and then centrifuge to collect supernatant for further analysis. Common transcription factors such as NFKB, p53 can be detected without sonication.

Validation Images & Assay Conditions

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