Western Blot Protocol

Western Blot Protocol

Materials:

Blocking Buller: 5% non-fat dry milk with TBST

TBST Washing Buffer

Procedures:

1. Electrophorese and transfer the gel to a membrane
2. Remove the membrane from the transfer unit.
3. Add enough blocking buffer to completely cover the membrane. Incubate while shaking for 1 hour.
4. Remove blot from blocking solution.
5. Dilute the primary antibody to the recommended dilution in 10mL of blocking buffer.
6. Incubate the blot with the primary antibody for 1 hour at room temperature.
7. Wash the blot 4 times (about 8 min each) in washing buffer with shaking.
8. Dilute 1μL anti-rabbit IgG-HRP conjugated secondary (or other appropriate secondary) in 10mL of blocking buffer to make a 1:10000 dilution.
9. Incubate blot with the secondary antibody for 1 hour at room temperature.
10. Wash the blot 4 times (about 8 min.) in washing buffer with shaking.
11. Discard washing buffer
12. Use enough TMB substrate solution to completely cover the membrane surface. Allow this to react for 2 minutes.