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