Wash cells with 10 mM Tris pH 8
French press
Slow speed spin
High speed spin
Resuspend in 10 mM Tris
Sonicate 2 x 15 sec to remove ß-lacatamases
Wash in 10 mM Tris
Resuspend in 10 mM Tris
Store at ?70ºC until needed.
Assay:
Incubate membranes with ~5 µCi 3H-penicillin at 25ºC for 10 minutes
Add 1000X cold penicillin
SDS-PAGE
Incubate in 1 M sodium salicylate for 30 minutes
Dry at 80ºC for 2 hours under vacuum
Expose to photographic film for 3-30 days.