Team:TU Darmstadt/Protocols/Purification of Periplasmatic Proteins

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Purification of Periplasmatic Proteins

Materials

Procedure

  1. 1 L cell culture centrifugate at 6500 rpm for 20 min
  2. Discard the supernatant
  3. Resolve the pellet in 20 mL ice cooled resuspension buffer
  4. Combine the solved pellets
  5. Shake for 30 min at 100 rpm on ice
  6. Centrifugate again at 6500 rpm for 10 min
  7. Repeat step 2-6
  8. Fill supernatant in a new flask
  9. Add 130 g ammonimsulfate
  10. Stir on ice until the ammoniumsulfate is solved
  11. Protein flocculates
  12. Centrifugate at 10000 rpm for 10 min
  13. Discard supernatant
  14. Leave the pellet for 5 min on ice
  15. Remove buffer with pipette
  16. Resolve the pellet in PBS
  17. Purificate the suspension with e.g. IMAC