Bio-Rad Immun-Blot® AP Colorimetric Kits - Manual (Page 12)

Problem Probable cause Recommended solution
Transfer of protein onto the
membrane was incomplete. Stain
the gel to assure transfer of
protein. Use prestained standards
to monitor transfer efficiency.
Consult the Trans-Blot, Trans-Blot
SD, or Mini Trans-Blot manual for
proper electrophoretic transfer
procedures. Refer to the Bio-Dot
or Bio-Dot SF manual for proper
dot blotting procedures.
First antibody Loss of activity may have oc-
is not specific curred during electrophoretic
or does not rec- transfer. Pre-test the reactivi-
ognize denat- ty of the antibody against
ured antigens both native and denatured
(common with antigen by a dot blot. Refer
monoclonals). to the Trans-Blot manual for the
transfer of native antigens.
Antigen is too Increase the amount of anti-
dilute. gen in the assay.
2. High back- Blocking is Increase the blocking step to
ground (refer insufficient. 60 minutes.
to experimen-
tal strategy
Gelatin block- Prepare and use fresh block-
section) ing solution is ing solution.
old.
Nitrocellulose Remove the membrane when
is left in the the reaction appears to be
color develop- complete. If precipitate in
ment solution the color development
too long. solution appears, decant the
solution and use fresh reagent.
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Problem Probable cause Recommended solution
Tween-20 may affect the
reactivity of some antibodies.
Eliminate Tween-20 from the
assay (except the wash and after
blocking).
Conjugate is Conjugate is improperly stored.
inactive (see Store at 4 °C. Avoid heat inac-
Section 3.1). tivation. Do not sub-ject to
repeated freeze-thaw cycles.
The concentration of conjugate is
non-saturating. Increase the
conjugate concentration used in
the assay.
Conjugate may be contaminated,
causing inactivation of the
antibody (of Protein A or Protein
G) or the enzyme.
Tap water may cause inactivation.
Use only distilled, deionized
water.
Azide is a potent inhibitor of
horseradish peroxidase. Use
thimerosal as a bacteriostat.
Little or no Tween-20 may wash bound
antigen is antigen from the membrane.
bound to the Eliminate Tween-20 from the
membrane. assay (except the wash after
blocking).
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