A western blot is a procedure used to detect specific proteins in a sample. The proteins are first separated by size using gel electrophoresis. They are then transferred to a membrane and incubated with antibodies that will bind to the protein of interest. The membrane is then washed and incubated with a secondary antibody that is conjugated to a detectable marker. The presence of the protein is then detected based on color or light emission.


  1. Run SDS-PAGE
  2. Transfer protein to nitrocellulose membrane
  3. Incubate with a blocking solution, e.g., milk to prevent non-specific binding to the membrane
  4. Pour off the blocking solution
  5. Add primary antibody and incubate
  6. Rinse to remove excess primary antibody
  7. Add secondary antibody conjugated with an enzyme or chromophore
  8. Rinse to remove excess secondary antibody
  9. Visualize by colorimetric, chemiluminescence, or fluorescence method, depending on the type of secondary antibody
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Courtney Simons
Courtney Simons
Dr. Simons is a food science educator. He earned his bachelor’s degree in food science, and Ph.D. in cereal science at North Dakota State University.