Protein Isolation / Concentration

Protein Isolation from Culture Medium

  1. Place 1 mL of culture media in 15 mL polypropylene tube.
  2. Add 2 Vol (=2 mL) of acetone, place on rocking table at 4°C for 60min.
  3. Spin at 12000 RPM (max. speed) for 10 min.
  4. Discard supernatant, redissolve in 75 µl H2O + 25 µl 4X Treatment buffer.
  5. Transfer to 1.5ml Eppendorf tube.
  6. Add 1/20 of 4X treatment buffer volume 2-mercaptoethanol (= 1.25 µl).
  7. Boil to brake disulfide bonds.

Acetone precipitation

  1. Place 1 mL of conditioned media in 15 mL polypropylene tube.
  2. Add 4 mL cold acetone.
  3. Place in freezer 1 - 2 hours.
  4. Spin at 3000 - 4000 rpm for 15 minutes.
  5. Resuspend in 50 µl H2O + 50 µl 2 x loading buffer or in 75 µl H2O and 25 µl 4 x loading buffer.
  6. Transfer to 1.5ml Eppendorf tube.
  7. Add 1/20 of 4X loading buffer volume Vol 2-mercaptoethanol (= 1.25 µl).
  8. Boil to brake disulfide bonds.

Protein Isolation from Cell Culture

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