New lysin purification protool
New lysin purification protool
1. Pellet 500ml cells (5min x 5000g). Resuspend in 20ml MBBM, add 20ul tetracyline
2. Add 150ul purified PBCV incubate 16hrs under fluorescent illumination with a 16 hr light/8 hr dark cycle at 25C on an orbital shaker (~95 rpm).
3. Collect cells and virus by centrifugation at 22,000g for 20 min at 20C.
4. Re-suspend cells in 20ml 25mM KOH, 50mM MOPS (pH 7) (wash).
5. Centrifuge 22,000g for 20 min at 20C, discard supernatant.
6. Suspend pellet in 1.5ml 25mM KOH, 50mM MOPS (pH 7) - add 1.5ml 10M LiCl, incubate 30 min at 37C
7. Centrifuge 22,000g for 20 min at 20C. Collect supernatant (avoid DNA)
This protocol assumes that salt extracts lysin from the virion rather than from cell walls. Yield seems to confirm this assumption.