Protein Solubility

Procedure

  1. Pellet 3 mL of culture in a 1.5 mL eppendorf tube.

  2. Rinse pellet twice with 1 mL of 10 mM Tris, pH 7.5, 0.15 M NaCl.

  3. Resuspend in 150 uL of 10 mM Tris, pH 7.5, 0.15 M NaCl.

  4. Subject to 10 pulses of sonication, with a minimum power setting and 80% duty cycle.

  5. Centrifuge sonicant at 14,000 G for 15 minutes.

  6. Remove supernatant.

  7. Wash pellet twice with 1 mL of 10 mM Tris, pH 7.5, 0.15 M NaCl.

  8. Resuspend in final volume of 150 μL.

  9. Remove a 5 uL aliquot of the pellet and supernatant.

  10. Mix with 5 μL of SDS buffer containing DTT.

  11. Heat for 15 minutes at 100 degrees in a thermocycler.

  12. Resolve protein using a 12.5% acrylamide gel, stain with Coomassie.

  13. Fix image gel using densiometry, allowing analysis of soluble and insoluble fraction.

Notes and Cautions

  • Can use internal standard of BSA at 2 mg/mL.