Penicillin Binding Protein Assay

Membrane prep:

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 ³H-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.