Penicillan Binding Protein Assay青霉素与细胞膜蛋白结合实验

1. Wash cells with 10 mM Tris pH 8
   
2. French press
   
3. Slow speed spin
   
4. High speed spin
   
5. Resuspend in 10 mM Tris
   
6. Sonicate 2 x 15 sec to remove ß-lacatamases
   
7. Wash in 10 mM Tris
   
8. Resuspend in 10 mM Tris
   
9. Store at ?70ºC until needed.

Assay:

1. Incubate membranes with ~5 µCi ³ H-penicillin at 25ºC for 10 minutes
   
2. Add 1000X cold penicillin
   
3. SDS-PAGE
   
4. Incubate in 1 M sodium salicylate for 30 minutes
   
5. Dry at 80ºC for 2 hours under vacuum
   
6. Expose to photographic film for 3-30 days.