No bands at all (blank membrane) (Western Blot)

Question

Accepted Answer

Membrane is completely blank — neither target protein nor housekeeping control shows any signal after ECL exposure. Common causes: 1) Transfer failed or membrane transfer was incomplete 2) Primary or secondary antibody is degraded / expired 3) ECL substrate has lost activity 4) Total protein loaded was too low 5) PVDF membrane was placed with the wrong side facing the gel Solutions: 1) Verify transfer with Ponceau S staining — confirm proteins did migrate to the membrane 2) Check antibody species, dilution, and expiration date; run a positive control 3) Use freshly prepared ECL substrate 4) Increase load to 20-40 μg total protein per lane, or verify quantification 5) PVDF: rough side faces the gel (down); for nitrocellulose, either side works