Yu:Western

=Western Blotting=

Immunoblotting

 * 2. Block nitrocellulose membrane on rocker for 20-30 minutes at room temperature with blocking buffer, or overnight at 4°C.
 * 3. Discard blocking buffer and add primary antibody. Incubate on rocker at room temperature for 1 hour, or overnight at 4°C.
 * 4. Draw off and save primary antibody for reuse. Rinse with 30-50mL PBST.
 * 5. Wash twice on rocker for 10 minutes in PBST.
 * 6. Make secondary antibody (2μL into 10mL Western Blocking Buffer) and incubate membrane on rocker at room temperature for 1 hour.
 * 7. Discard secondary antibody. Rinse with 30-50mL PBST
 * 8. Wash twice on rocker for 10 minutes in PBST.
 * 9. Wash with distilled H2O for 10 minutes.
 * 10. Develop and image.
 * }
 * 7. Discard secondary antibody. Rinse with 30-50mL PBST
 * 8. Wash twice on rocker for 10 minutes in PBST.
 * 9. Wash with distilled H2O for 10 minutes.
 * 10. Develop and image.
 * }
 * 9. Wash with distilled H2O for 10 minutes.
 * 10. Develop and image.
 * }
 * }

Stripping Buffer
Procedure


 * 1) Wash Membrane for 15-20 min in 1x PBST at room temperature to wash away developing solution
 * 2) Incubate membrane in 50 mL of stripping buffer on a shaking platform for 30 min at 50°C
 * 3) Wash Membrane for 15-20 min in 1x PBST at room temperature
 * 4) Block and reprobe membrane with desired antibody