Team:TU Darmstadt/Protocols/Purification of Periplasmatic Proteins
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Latest revision as of 21:37, 26 September 2012
Purification of Periplasmatic Proteins
Materials
- Resuspension buffer
- 10 mM Tris buffer, pH 8.8
- ice
- Ammoniumsulfate
- Centrifuge
Procedure
- 1 L cell culture centrifugate at 6500 rpm for 20 min
- Discard the supernatant
- Resolve the pellet in 20 mL ice cooled resuspension buffer
- Combine the solved pellets
- Shake for 30 min at 100 rpm on ice
- Centrifugate again at 6500 rpm for 10 min
- Repeat step 2-6
- Fill supernatant in a new flask
- Add 130 g ammonimsulfate
- Stir on ice until the ammoniumsulfate is solved
- Protein flocculates
- Centrifugate at 10000 rpm for 10 min
- Discard supernatant
- Leave the pellet for 5 min on ice
- Remove buffer with pipette
- Resolve the pellet in PBS
- Purificate the suspension with e.g. IMAC