Bio-Rad Trans-Blot® Cell - Manual (Page 22)

No Reaction or Weak Signal
1. The sample load was insufficient.
• Increase the amount of protein applied. Concentration of the sample
prior to loading may be necessary. Use a more sensitive assay system
(see Table 2.10).
2. Insufficient antigen binding to the membrane is occurring.
• Stain the gel after transfer or use prestained or Kaleidoscope standards
to assess transfer efficiency. See the previous section for suggestions on
improving transfer related problems.
3. Antigen denaturation is occurring during electrophoresis or transfer.
Antibodies, especially monoclonals, may not recognize denatured antigens.
• Electrophorese and transfer proteins under native conditions. Use the
Super Cooling Coil and a refrigerated recirculating bath to transfer heat-
sensitive proteins.
4. Primary or secondary antibodies may be inactive or non-saturating.
• Store the reagents at recommended conditions. Avoid repeated freeze-
thaw cycles, bacterial contamination, or heat inactivation.
• Detergents may affect the activity of some antibodies. Eliminate them
from the assay, except for the wash after blocking.
• If the antibody titer is too low, optimize the concentration using a
dot-blot experiment.
• Increase the antibody incubation times.
5. The enzyme conjugate is inactive or non-saturating.
• Test the reagent for activity (see below).
• Store the reagents at recommended conditions. Avoid repeated freeze-
thaw cycles, bacterial contamination, or heat inactivation.
• Sodium azide is a potent inhibitor of horseradish peroxidase. Use
Thimerosal as a bacteriostat.
• Impure water may cause inactivation of the enzyme. Use only distilled,
deionized water.
• If the conjugate concentration is too low, optimize using a dot-blot
experiment.
6. Color development reagent is inactive.
• Test the reagent for activity (see below) and remake if necessary.
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. The color reaction should devel-
op immediately. If color fails to develop within a few minutes, the color
development solution is inactive. Make up a fresh working solution and
repeat the color development assay.
2. Activity test for the conjugate solution.
• Combine 1.0 ml of the color development solution tested above 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 min-
utes, the conjugate solution is suspect. Repeat the procedure with a
freshly prepared dilution of conjugate.
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