Western 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