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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.
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.
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.
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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