Bio-Rad Immun-Blot® AP Colorimetric Kits - Manual (Page 8)

reactions. At 0.05%, Tween-20 will not disrupt binding of primary
antibodies to antigens or antigens to nitrocellulose, but will
optimize detection sensitivity by eliminating non-specific reactions.
Alternative detergents, or concentrations of Tween-20 other than
0.05% should not be substituted. The wash between blocking the
nitrocellulose with TBS - 3% gelatin and probing with first
antibody is essential and should not be altered.
8. Color development reagents - Immun-Blot assay kits are provided
with one of two color development reagent systems. All color
development reagents are ready to use after preparing a stock
solution of AP or HRP color development buffer.
9. Technical Service - Contact Bio-Rad Laboratories Technical
Services Group in Hercules, California, toll free 1-800-4BIORAD,
or your local Bio-Rad representative, if you require assistance.
2.2 Detailed Assay Procedure
Note: Before beginning, read through the entire procedure.
1. Antigen applications - Apply antigen to the membrane surface
using one of the three basic methods described below. A small
amount of known antigen or primary antibody dotted on one corner
of the membrane prior to blocking will develop color if the
procedure is successful. This is an excellent check on the operation
of the assay and will help you to gauge the rate of color
development.
a. Dot-blotting - Cut the nitrocellulose sheet to appropriate size
(i.e. 0.9 x 9.2 cm strips, 3 x 5 cm rectangles, or Petri dish
circles). It is advisable to draw a grid on the membrane with a
pencil. Typical grids consist of 1 x 1 cm squares. Next, the dry
nitrocellulose is wetted by slowly sliding the membrane at a 45°
angle into TBS. Remove the thoroughly wetted membrane from
the TBS and dry it on filter paper for approximately 5 minutes.
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2. Temperature - All steps are performed at room temperature (22-
25 °C) unless indicated otherwise in the instructions. If a lower
assay temperature is required, it is advisable to double the
incubation and wash times for each 10 °C decrease in temperature.
In low temperature incubations, it may be necessary to substitute
BSA for gelatin.
3. Wash Purity - Poor quality water can contain inhibitors of
enzymatic color development. Use only deionized, distilled water
to prepare all solutions.
4. First antibody - Generally, when serum or tissue culture
supernatants are the source of primary antibody, a 1:100-1:1,000
dilution of the primary antibody in antibody buffer is used for
detection of antigens on the membrane surface. For
chromatographically purified monospecific antibodies, a 1:500-
1:10,000 dilution in antibody buffer is used for antigen detection. A
1:1,000-1:100,000 dilution is used when ascites fluid is the source
of antibody. Optimal dilution factors must be determined
experimentally. The optimal antibody concentration is usually
considered the greatest dilution of antibody reagent still resulting in
a strong positive signal without membrane background or
nonspecific reactions.
5. Conjugates - The conjugates supplied by Bio-Rad should be used
in the concentrations indicated in Section 1.5. Using a conjugate at
higher concentrations may result in an overall increase in
background without any increase in detection sensitivity.
6. Washes and incubations - Continuous gentle agitation should be
used during all reactions. For best results, an orbital shaker should
be employed to maintain a uniform exposure of the membrane to
the solution.
7. Addition of detergents - Tween-20 is essential in washing to
eliminate overall background and non-specific hydrophobic
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