Which laboratory method is used to separate proteins in the Western Blot?

• A. Mass spectrometry
• B. Chromatography
• C. Ultracentrifugation
• D. Gel electrophoresis

Answer: (D)

Separating proteins in a Western blot relies on gel electrophoresis, typically SDS-PAGE. Proteins are denatured and coated with SDS to give them a uniform negative charge, then loaded into a polyacrylamide gel. An electric field drives the proteins through the gel, and smaller proteins move faster, creating size-based separation. This separated pattern is what gets transferred to a membrane for antibody detection. Other methods—mass spectrometry, chromatography, or ultracentrifugation—serve different purposes or don’t provide the same size-based separation step used in the Western blot workflow.