Team:TU Darmstadt/Protocols/Purification of Periplasmatic Proteins

Purification of Periplasmatic Proteins

Materials

• Resuspension buffer
• 10 mM Tris buffer, pH 8.8
• ice
• Ammoniumsulfate
• Centrifuge

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