Bio-Rad Immun-Blot® Opti-4CN™ Colorimetric Kits - Manual (Page 10)

This approach entails two rounds of amplification, so
background can be a significant problem and it may not be
trivial to work out the best conditions. Background is
affected by the dilution of primary antibody, secondary
antibody, the BAR and the streptavidin-HRP. It may be
necessary to further dilute one or more of these four
reagents.
A brief protocol is outlined below.
Blotting and amplification.
1. Transfer protein to nitrocellulose membrane by elec-
trophoretic blotting, dot blotting or microfiltration.
Allow membrane to air dry.
2. Wet membrane in PBST and then wash 2x for 5 min-
utes each time in PBST.
3. Block membrane in 3% blocker for 1 hour.
4. Wash 2x with PBST for 3–5 minutes.
5. Incubate in appropriately diluted primary antibody
for one hour.
6. Wash 2x with PBST for 5 minutes each time.
7. Incubate in 1:3,000–1:10,000 dilution of biotinylated goat
anti-rabbit IgG secondary antibody (catalog number
170-6401) for 1 hour.
8. Wash 2x with PBST for 5 minutes each time.
9. Incubate in 1:1,000 dilution of streptavidin-HRP for
30 minutes.
15
12. Wash. Pour off the streptavidin-HRP solution and add
PBST. Wash the membrane for 5 minutes with gentle
agitation. Repeat the wash step with fresh PBST.
Colorimetric Detection
13. Prepare 0.25 ml of Opti-4CN diluent solution per cm 2
of membrane by first mixing 1 part Opti-4CN diluent
concentrate with 9 parts ddH
2
O. For each 10 ml of
diluent, add 0.2 ml of Opti-4CN substrate and mix
well. The solution will become cloudy upon mixing.
14. Pour the PBST off the membrane and add the pre-
pared substrate solution. Incubate for 5–30 minutes
or until the desired level of signal is attained.
15. Pour off the substrate and wash the membrane in
ddH
2
O for 15 minutes.
16. Dry the membrane and store or document.
Section 3
Alternative Protocol
(For kits 170-8238/39/40)
An alternative, and potentially more sensitive ampli-
fication, may be accomplished by substituting a biotiny-
lated secondary antibody for the HRP-linked secondary
antibody. There is an additional incubation in streptavidin-
HRP that precedes incubation in the BAR solution. The kit
has sufficient streptavidin-HRP to carry out detection over
2,500 cm
2 of membrane, even if you use a biotinylated
secondary antibody.
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