Bio-Rad Immun-Blot® AP Colorimetric Kits - Manual (Page 11)

3.2 Troubleshooting Guide
Problem Probable cause Recommended solution
1. No reaction or Color develop- Color development reagents
weak color ment solution must be stored at the proper
development is inactive (see temperature (see Section 1.3).
Section 3.1).
HRP color development solution
will precipitate out of solution if
kept at 0-4 °C, and detection
sensitivities may be decreased.
Warm the HRP color development
buffer to RT prior to addition of the
color reagent solutions. HRP color
reagents A and B should be kept at
their proper storage temperature
prior to mixing to maximize their
activity and prolong their shelf life.
Avoid bacterial contamination of
the color development buffers by
storage at 4 °C.
Tap water can inactivate the color
development solution. Use only
distilled, deionized water to
prepare the solutions.
First antibody Antibody is improperly stored.
solution is in- Avoid bacterial contamination,
active or non- heat inactivation, and repeated
saturating (see freeze-thaw cycles.
Section 3.1).
Antibody titer is too low. Increase
the concentration of antibody used
in the assay.
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Section 3
Troubleshooting
3.1 Tests for Monitoring Reagent Activity
1. Activity test for the color development solution.
Combine 1.0 ml of the color development solution with 10 µl of
full strength second antibody conjugate or Protein A or Protein G
conjugate. The color reaction should develop immediately. If color
fails to develop within a few minutes, the color development
solution is inactive. Make up fresh working solution and repeat the
color development assay.
2. Enzyme activity test for the conjugate solution.
Combine 1.0 ml of the color development solution (tested in step 1)
and 1.0 ml of the 1:3,000 dilution conjugate solution. A light blue
tinge should develop within 15 minutes. If color fails to develop
within 25 minutes, the conjugate solution is suspect. Repeat the
procedure with a freshly prepared dilution of conjugate.
3. Activity test for the first antibody solution.
Use a RID, Ouchterlony immunodiffusion, precipitation, or ELISA
test to determine reactivity of the antibody with the antigen. If
possible, repeat the Immun-Blot procedure with a more
concentrated first antibody solution.
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