Far Western Blot Protocol

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Far Western Blot

1. Perform SDS-PAGE and Western Blot transfer as normally (SDS Gel Electrophoresis and Western Blot Protocol)
2, Block with blocking agents (eg milk or BSA); may want to block longer than with a normal western blot, or with a higher concentration of blocking agent
3. Incubate with ligand in blocking buffer for 2 hours or longer. (May want to do this at 4 degrees C if high nonspecific binding occurs.)
4. Wash with TBS-Tween
5. Perform the Western blot as normally with primary antibody against the ligand, and then subsequent secondary antibody and development
6. As a control, may want to perform a blot without the ligand and just with primary and secondary antibody incubations.