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Total Protein Extraction by RIPA

Protein extraction from tissues and cultured cells is the first step for many biochemical and analytical techniques such as protein purification, western blotting, as well as mass spectrometry. There is still no universal set of method or regent that is optimal for protein extraction due to the chemical/physical heterogeneity of proteins and their sample sources. Some factors should be taken into account when handling proteins such as sample types, subcellular locations of the protein, the compatibility of the protein as well as downstream applications. Here we provide a protocol for total protein extraction based on RIPA which is widely used.

View more information about total protein extraction by TRIzol.

Notes:

  • All reagents and instruments must be pre-cold to reduce protein degradation.
  • Extract total protein quickly and efficiently.
  • Add 10 µL sodium orthovanadate solution and 10 µL protease inhibitor cocktail solution per 1 mL of RIPA lysis buffer before use to prevent proteolysis and maintain phosphorylation of proteins.
  • Protein production should be store at -80°C.

Reagents:

  • PBS: Dissolve 8 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4 and 0.2 g KH2PO4 in 800 mL distilled water. Adjust the PH to 7.4 with HCl and final volume to 1 liter with additional distilled H2O.
  • RIPA lysis buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1mM EDTA, 1% NP-40, 1% Na-deoxycholate, 0.1% SDS, sterile-filtered.
  • Protease and phosphatase inhibitors: Protease inhibitor cocktail and sodium orthovanadate.

Equipment:

  • Plastic cell scraper for adherent cells
  • Microcentrifuge tube
  • Round-bottom microcentrifuge tube
  • Centrifuge
  • Electric homogenizer
  • Suction and Pasteur pipette
  • Scissors and scalpel blade
  • Pipettes and pipettors

Procedures:

Protocol A: Total Protein Extraction from Cell Suspension

1. Collect cells by centrifugation the suspension at 500 x g for 5 minutes and aspirate the culture media carefully.

2. Wash cells with ice-cold PBS. Centrifuge at 500 x g for 5 minutes at 4°C and aspirate the supernatant.

3. Repeat Step 2 twice.

4. Add 1 mL ice-cold RIPA lysis buffer for 1 x 107 cells and agitate the contents in microcentrifuge tube for 20 min at 4°C.

5. Centrifuge at 13,000 x g for 20 min at 4°C.

6. Carefully collect the supernatant containing the soluble protein in a new tube kept on the ice for further analysis. Discard the pellet.

Protocol B: Total Protein Extraction from Adherent Cells

1. Aspirate the culture media from adherent cells slowly by suction and Pasteur pipette.

2. Carefully wash adherent cells with ice-cold PBS, rock gently and aspirate the PBS to remove residual medium.

Note: Slowly add a volume of ice-cold PBS in order to not dislodge cells.

3. Repeat Step 2 twice.

4. Add 1 mL ice-cold RIPA lysis buffer for 1 x 107 cells. Scrape the plate using a cold plastic cell scraper to lyses residual cells then gently transfer cell lysate to a 1.5 mL microcentrifuge tube.

5. Agitate the contents in microcentrifuge tube for 20 min at 4°C.

6. Centrifuge at 13,000 x g for 20 min at 4°C.

7. Carefully collect the supernatant containing the soluble protein in a new tube kept on the ice for further analysis. Discard the pellet.

Protocol C: Total Protein Extraction from Tissue

1. Weigh a certain amount of tissues and cut into pieces on ice.

2. Transfer tissues to a round-bottom microcentrifuge tube and snap-freeze by immersing in liquid nitrogen.

3. Add 300 µL ice-cold RIPA lysis for 5 mg tissue and homogenize with an electric homogenizer on ice.

Note: sonication is also recommended.

4. Wash the blade twice with additional 400 µL ice-cold RIPA lysis buffer.

5. Agitate the contents for 2 h at 4°C.

6. Centrifuge at 13,000 x g for 20 minutes at 4°C.

7. Carefully collect the supernatant containing the soluble protein in a new tube kept on the ice for further analysis. Discard the pellet.

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