Bio-Rad Immun-Blot® AP Colorimetric Kits - Manual (Page 9)

Overnight incubation may be preferred, since longer incubation
periods may increase the sensitivity of detection.
5. Washes - Remove the unbound first antibody by washing the
membrane in TTBS for 5 minutes at RT. Decant the wash solution
and repeat the wash with another portion of TTBS for 5 minutes at
RT. Protein G-HRP assays, these washes should be done in TCBS
instead of TTBS. The optimal binding pH of Protein G is 5.5.
Therefore, the Protein G-HRP binding step should be carried out in
the citrate buffer.
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6. Conjugate binding step - Decant the TTBS and add the second
antibody solution, Protein A-HRP conjugate solution, or Protein G-
HRP conjugate solution, and incubate 30 minutes to 2 hours using
gently agitation at RT. The optimum conditions of dilution and
incubation time must be determined experimentally.
7. Final washes - Remove the conjugate solution * , and wash the
membrane in TTBS for 5 minutes with gently agitation at RT.
Decant the solution and wash with another portion of TTBS for 5
minutes at RT. Just prior to color development, wash the membrane
in TBS for 5 minutes at RT with gentle agitation to remove residual
Tween-20 from the membrane surface.
* Save used conjugate solution for testing in case results are unsatisfactory. See
Section 3.1.
2.3 Detailed Color Development Procedure
Procedure for Alkaline Phosphatase Blots
1. Developer preparation - Immediately before use, add 1.0 ml of
AP color reagent A and 1.0 ml of AP color reagent B to 100 ml 1x
of AP color development buffer at RT. This solution can be stored
at 4 °C overnight, but prompt use is recommended.
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Apply sample antigen to each grid square using a syringe or a
variable pipette, by displacing 1 µl of sample to the tip of the
syringe or pipette as a drop and gently touching it to the surface
of the nitrocellulose membrane. If the antigen sample is very
dilute, it is possible to apply successive 1 µl doses at the same
spot by letting the previous sample application dry completely
before adding an additional dose. In all cases, the nitrocellulose
membrane should be allowed to dry completely before
proceeding to the blocking step.
b. Electrophoretic blotting - The anitgens of interest are
electrophoretically transferred to the membrane from a gel
support (i.e. SDS-PAGE gel, IEF gels, or native gels) using the
Trans-Blot
® , Mini Trans-Blot, or Trans-Blot SD cell. If desired,
cut the wet nitrocellulose membrane into 0.6-0.8 cm wide strips.
Immerse the strips or the entire sheet in TBS proceeding to the
blocking step.
c. Microfiltration blotting - The Immun-Blot assay kits can easily
be adapted for use in the Bio-Dot or Bio-Dot SF apparatus.
These instruments allow rapid, reproducible applications of up to
96 samples on one membrane sheet. All applications and
washes, except color development, are carried out in the
apparatus.
2. Blocking step - After the antigen is applied, using one of the above
methods, immerse the membrane, at a 45° angle, into the blocking
solution. Gently agitate the solution using an orbital shaker platform
and incubate for 30 minutes to 1 hour at room temperature (RT).
3. Wash - Decant the blocking solution and add TTBS to the
membrane. Wash for 5-10 minutes with gently agitation at RT.
4. First antibody - Decent the TTBS and add first antibody solution
to the membrane. Incubate 1 to 2 hours with gently agitation.
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