Bio-Rad Immun-Blot® Opti-4CN™ Colorimetric Kits - Manual (Page 8)

Probable Cause Recommended Solution
2. Color development solution 2. Follow activity test proce-
is inactive. dure for color development
reagents (see Section 3.1).
a. Reagent improperly stored. a. Store desiccated at
Wetness of a regent or an -20 °C and away from
obvious color change may light.
be symptomatic.
b. Light inactivation of color b. Mix solution out of direct
development reagent or light and use fresh,
H
2
O
2
. stabilized H
2
O
2
.
c. Reagent precipitated out c. Mix solution at room
of solution. temperature See
Section 1.4. Methanol can
also react with Tween -20
to produce a precipitate.
This will not interfere
with color development.
3. Avidin-HRP conjugate solu- 3. Follow activity test for
tion is inactive. conjugate (see Section 3.1).
a. Avidin-HRP conjugate im- a. Store at 0–4 °C or freeze
properly stored. aliquots to avoid repeated
freeze-thaw cycles.
4. Inactive NHS-biotin solution.
a. NHS-biotin reagent impro- a. Store the NSH-biotin
perly stored. reagent at 20 °C. Equili-
brate reagent to room
temperature before open-
ing. Use a sterile syringe
to remove reagent to pre-
vent contamination and
condensation of water
inside the vial.
b. NHS-biotin reagent is b. The NHS-biotin reagent
hydrolyzed. hydrolyzed in aqueous
solutions. NHS-biotin
reagent should be added
last to the BT solutions
containing the membrane.
11
2. Prepare five test tubes containing the following solutions:
a. Solution 1: 1.0 ml of 0.1 N NaOH + 10 µl DMF
b. Solutions 2–4: 1.0 ml of 0.1 N NaOH + 10 µl NHS-biotin
reagent
c. Solution 5: 1.0 ml of 0.1N HCl + 10 µl NHS-biotin
reagent
3. Heat solutions 1–4 to 100 °C for 10 minutes. Do not heat
solution 5.
4. Add 0.1 ml of 1.0 N HCl and 100 µl of 0.05 M Fe(NO
3
)
3
in
0.5 N HCl to solutions 1–5. Mix thoroughly.
5. Measure the absorbance of each solution at 500 nm. Subtract
the absorbance value of solution 1 from that of solutions 2–5.
6. Solutions 2–4 should be a pale red color. Determine the aver-
age absorbance value for solutions 2–4. (The absolute
absorbance values may vary depending upon the age and qual-
ity of the Fe(NO
3
)
3
.)
7. Solution 5 should be pale yellow to colorless. Presence of a red
color indicates hydrolysis of the NHS-biotin reagent. If the
absorbance of solution 5 is greater than 10% of the average
absorbance of solutions 2–4, the NHS-biotin reagent is suspect.
3.2 Troubleshooting Guide
A. No reaction or weak color development
Probable Cause Recommended Solution
1. Enzyme inactivated.
a. Tap water or water deio- a. Use distilled, deionized
nized by polystyrene resin H
2
O. Other reagents
may inactivate the enzyme. should be of the highest
purity.
b. Azide is a potent inhibitor b. Do not use sodium azide
of the enzyme. in any of the solutions, If
a bacteriostat is neces-
sary, use thimerosal
(Merthiolate
® ) at a 0.01%
concentration.
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