Bio-Rad Clarity™ Western ECL Substrate User Manual
Page 10
6 Technical Support: 1-800-424-6723 • www.bio-rad.com
4. Incubate the membrane in diluted primary antibody solution
for 1 hr with continuous agitation.
•
Incubation in primary antibody may be carried out
overnight at 4°C
5. Wash the blot in wash buffer 5 times for 5 min each with
continuous agitation.
6. Incubate the blot in diluted secondary antibody solution for
1 hr with continuous agitation.
7. Wash the blot in wash buffer 6 times for 5 min each with
continuous agitation.
Chemiluminescent Development
1. Keep the membrane moist in wash buffer as you prepare
the substrate mixture.
•
Do not allow the membrane to dry out during the
subsequent steps
2. Mix substrate kit components in a 1:1 ratio. Prepare 0.1 ml
of solution/cm
2
of membrane.
•
For a mini-sized membrane (7 x 8.5 cm), 7 ml of
solution is sufficient
•
For a midi-sized membrane (8.5 x 13.5 cm), 12 ml of
solution is sufficient
3. Place the membrane, protein side up, on a clear surface.
•
Add substrate to the blot and incubate for 5 min
•
Ensure the surface of the blot is completely covered
with substrate with no air bubbles