Bio-Rad Immun-Blot® Opti-4CN™ Colorimetric Kits - Manual (Page 7)

1 second exposure in the reflectance mode. Film should be
developed 1 minute.
Dry nitrocellulose or nylon membranes between sheets of fil-
ter paper. Store between polyester sheets. Protect from light to
minimize fading.
Section 3
Troubleshooting
3.1 Tests for Monitoring Reagent Activity
A. Activity test for the color development solution.
Combine 1.0 ml of the color development solution with 10 µl
of Avidin-HRP conjugate. If the color does not develop in
5 minutes, either the color development reagent or H
2
O
2
is at
fault. Add 1 µl of fresh 30% H
2
O
2
. If color does not develop,
the color development reagent is suspect. If color develops,
H
2
O
2
is at fault.
B. Enzyme activity test for the Avidin-HRP solution.
Combine 1.0 ml of the color development solution (tested in 1
above) and 1.0 ml of the Avidin-HRP solution (1:1,000). If
color fails to develop with 5 minutes, the conjugate solution is
suspect. Repeat the procedure beginning at Step 6 with a
freshly prepared dilution of Avidin-HRP.
C. Activity test for the NHS-Biotin reagent.
1. Reagent required
a. 0.1 N HCl
b. 1.0 N HCl
c. 0.05 M Fe(NO
3
)
3
in 0.5 N HCl
d. 0.1 N NaOH
e. Dimethylformamide (DMF)
9
3. Discard the wash solution and replace it with 100 ml of fresh
BT solution. While agitating the incubation vessel, add 200 µl
of NHS-biotin. Incubate the membrane for 15 minutes with
constant agitation. Do not prepare BT solution containing
NHS-biotin before use, as the biotin reagent is hydrolyzed
in aqueous solution.
Note: To prevent hygroscopic accumulation of water in the
NHS-biotin reagent, equilibrate the vial to room temperature
before use and remove reagent with a sterile syringe.
4. Wash the membrane in 100 ml of BT for 5 minutes. Repeat.
5. Wash the membrane in 100 ml of TTBS for 5 minutes.
Repeat.
6. Prepare the Avidin-HRP solution as described in Section 1.4.
Reserve a 1.0 ml sample for troubleshooting should a problem
arise. Incubate the membrane in the Avidin-HRP solution for
1 hour with agitation.
7. Wash the membrane in 100 ml of TTBS for 5 minutes,
Repeat.
8. Wash the membrane in TBS for 5 minutes. Repeat.
9. Prepare the HRP color development solution immediately
before use as described in Section 1.4. To prevent undesirable
precipitates, solution (b) must be at room temperature before
addition of solution (a). Reserve a 2.0 ml sample for trou-
bleshooting should a problem arise. Immerse the nitrocellu-
lose or nylon membrane in the color development solution and
begin agitation. Protein concentrations greater than 250 ng
will immediately become visible as purple bands or dots.
Develop color for 15–30 minutes on nitrocellulose mem-
branes or 3–5 minutes on Zeta-Probe membranes (when work-
ing with nylon membranes, extended periods of color
development will result in increased background). If a heavy
precipitate forms in the color development solution, a fresh
solution should be prepared and used immediately.
10. Stop color development by immersing the membrane in dis-
tilled water for 10 minutes. Change the water at least once to
remove remaining color development solution. Take pho-
tographs while the membrane is wet to enhance the purple
bands or dots. Acceptable photographs can be produced by
using Polaroid Type 108, Polacolor 2 Land film, at f8 and
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