Haynes:ProteinExtraction

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Protein Extraction: active protein from cultured mammalian cells

by Karmella Haynes, 2014; adapted from G-Bioscience Application Note #3 "Protein Extraction and Lysis Buffers (PE LB)

Principle: Use a buffer that is formulated for gentle extraction of biologically active proteins from mammalian cells. Proteins can be column-purified and/or used for activity assays or native gel electrophoresis.

MATERIALS

• Protein extraction buffer (e.g., Mammalian Cell-PE LB™, G-Biosciences #786-180)
• Protease inhibitor cocktail (e.g., ProteaseArrest™ 100x, G-Biosciences #786-108)
• 5x10⁶ cells, approximately 1/2 the amount of adherent U2OS or Hela cells grown in a T-75 flask or 10 cm round culture dish at 90% - 100% confluency.

PROCEDURE

Grow and harvest the cells (do this part under sterile conditions in the TC room)

1. Grow adherent cells in a T-75 or 10 cm round culture dish to 90% - 100% confluency.
2. Harvest the cells using a standard trypsinization procedure.
3. After the cells are suspended in growth medium to a final volume of 10 mL, transfer 5 mL of cells into two 15 mL conicals.
4. Spin the cells for 3 min. at 1000 rpm at room temp.
5. Aspirate off the growth medium and leave ~100 uL covering the pellets.
6. Flick each tube to break up the pellets.
7. Bring the cells from the TC room to your bench

Lyse the cells

1. Label two clean 1.5 mL microcentrifuge tubes.
2. Add 500 μL of protein extraction buffer to each tube.
3. Add the appropriate amount of protease inhibitor cocktail to the protein extraction buffer in each tube (e.g., 5μL of 100x inhibitors). Close the caps and invert to mix.
4. Add one protein extraction buffer + inhibitor mix to one cell sample in the 15 mL conical. Pipette up and down. Transfer the lysed cells back to the 1.5 mL tube. Using a fresh tip, repeat this step for the other sample.
5. Vortex each lysed cell sample for 1 min.
6. Go to the cold room (4°C) and centrifuge the samples for 5 min. at top speed.
7. Bring the samples back to the bench. Label two new 1.5 mL tubes.
8. Using a micropipette, transfer the clear supernatant into each new tube. Avoid the thick, viscous pellet at the bottom. If this pellet is dsirupted, pipette the lysate back into the tube and repeat the 4°C centrifugation step.