GENERAL GUIDELINES FOR WESTERN BLOT

NOTE:
To achieve the best results, optimization of many conditions must be done.  Assay conditions such as antibody and antigen working concentrations, incubation temperatures, and incubation times must be evaluated.  For optimal results, each laboratory should determine the best working conditions for their specific assay.

1. Transfer proteins to nitrocellulose membrane using a standard Western blot transfer protocol.
2. Block the membrane with 2% BSA or 3% non-fat dry milk in PBS pH 7.4-0.1% Tween20.  Incubate with gentle shaking at room temperature for 2 hours.
3. Wash the membrane with gentle shaking for 3 x 5 minutes with PBS-0.1% Tween20.
4. Add antibody diluted in blocking solution to the membrane.  Use a volume sufficient to completely cover the membrane.  Incubate with gentle shaking at room temperature for 1-4 hours.
5. Wash the membrane with gentle shaking for 3 x 5 minutes with PBS-0.1% Tween20.
6. Add HRP-labeled secondary antibody diluted in blocking solution.  Incubate with gentle shaking at room temperature for 1 hour.
7. Wash the membrane with gentle shaking for 3 x 5 minutes with PBS-0.1% Tween20.
8. Develop using chromogen or ECL substrate.