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Functional Genomics of Plant Pathosystems

Western procedure

Procedure

  1. Run gel at 100 Volt in Running buffer for 30 minutes or until proteins have entered resolving gel.
  2. Increase voltage to 120-150 Volt until good separation is obtained for your protein of interest.
  3. Make the following sandwich (pre-wet with transfer buffer):
  4. 2 Whatman paper, place your gel (no need to rinse or soak it), place your nylon membrane (or pre-wetted PVDF), 2 more Whatman. Close sandwich and place in transfer unit. Run in cold transfer buffer with cold pack for 1 hour @ 100 Volt (not higher).
  5. Carefully remove membrane from sandwich and immerse in TBS for 5 minutes.
  6. Block for 1 hour in blocking buffer.
  7. Add new blocking buffer + your primary antibody at the appropriate concentration.
  8. Incubate 1 hour.
  9. Rinse 3 time 5-15 minutes with wash buffer
  10. Add new blocking buffer + your secondary antibody at the appropriate concentration.
  11. Incubate 1 hour.
  12. Rinse 3 time 5-15 minutes with wash buffer
  13. Use detection reagent