Bio-Rad Clarity™ Western ECL Substrate - Manual (Page 9)

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Detailed Assay Procedure
Example Western Protocol
Materials
• PVDF, LF PVDF, or nitrocellulose membrane with transferred
proteins
• Blocking buffer (Tris-buffered saline (TBS) or phosphate-
buffered saline (PBS) with 0.05% Tween-20 and 1–6% of
a blocking reagent, typically BSA, gelatin, casein, or nonfat
dry milk
• Wash buffer (TBS or PBS with 0.05% Tween-20)
• Primary antibody, diluted in blocking buffer
• HRP-conjugated secondary reagent, such as goat
anti-rabbit or goat anti-mouse conjugated HRP, diluted in
wash buffer
Immunodetection
1. Wash buffer volumes should be at least 20 ml for mini blots
and 100 ml for midi blots. Block and antibody solution
volumes should be at least 10 ml for mini blots and 25 ml for
midi blots.
2. Equilibrate membrane with transferred proteins in wash
buffer for 3 minutes. Dried PVDF and LF PVDF membranes
should be briefly re-wet in methanol prior to equilibration in
wash buffer.
3. Incubate the membrane, protein side up, in blocking buffer
for 1 hr with continuous agitation.