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

来源:来源网络 2007-07-04 22:44

  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.

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