PenicillanBindingProteinAssay青霉素與細胞膜蛋白結合實驗

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 ?-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.

7.