Bio-Rad Immun-Blot® AP Colorimetric Kits - Manual (Page 7)

Second antibody Prepare the second antibody solution by dissolving 33 µl
conjugate solution of the antibody conjugate in 100 ml of antibody buffer,
or Protein A-HRP if applicable.
conjugate solution
Protein G-HRP Dissolve 33 µl of the conjugate solution in 100 ml of
conjugate solution 1% gelatin in TCBS, if applicable.
Note: If a bacteriostat is needed, 0.01% thimerosal should be used.
Sodium azide is potent inhibitor of horseradish peroxidase.
Section 2
Immun-Blot Assay
2.1 Experimental Strategy and General
Recommendations
1. Background - Three types of background are common to immune
blot detection.
a. High membrane coloration - High membrane backgrounds
usually result when the blocking period is too short, when
Tween-20 is absent from the appropriate buffers and washes, or
when excessive amount of conjugate are used.
b. Non-specific antibody binding - Usually evidenced by extra
banding or high coloration in the separate lanes. This
background is usually due to impure or cross-reactive
antibodies, incubations with excessive antibody concentrations,
or an absence of Tween-20 from the appropriate buffers and
washes.
c. Non-specific conjugate binding - This background is
evidenced by band development in the absence of first antibody.
It results when conjugate is used in excess, or when Tween-20
is absent from the appropriate buffers and washes.
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Since the AP color development buffer is better stored
for longer periods as a concentrate, make as much 1x
solution as is practical and needed for the planned
experiments.
Wash solution (20 mM Tris, 500 mM NaCl, 0.05% Tween-20, pH 7.5)
(TTBS) Add 350 µl of Tween-20 to 700 ml to 1x TBS. Label
this bottle “TTBS.”
For the Protein G-HRP assay, make 400 ml of TTBS
instead, using 200 µl of Tween-20. Also prepare 300 ml
of TCBS by adding 150 µl of Tween-20 to 300 ml of
CBS. Label this bottle “TCBS.”
Blocking solution (3% gelatin - TBS)
Add 3.0 g of gelatin to 100 ml of TBS and heat to 50 °C,
with stirring, until dissolved.
A microwave oven will quickly solubilize the gelatin,
but do not heat above 65 °C. Label this solution
“Blocking solution - 3% gelatin in TBS.”
Antibody Buffer (1% gelatin - TTBS)
Add 2.0 g of gelatin to 200 ml of TTBS and heat, with
stirring to 50 °C until dissolved. Again, you can use a
microwave oven. Label this solution “Antibody buffer -
1% gelatin in TTBS.” Store at 4 °C.
For the Protein G-HRP assay, prepare only 100 ml of
1% gelatin in TTBS and prepare 100 ml of 1% gelatin
in TCBS.
First antibody Dissolve the first antibody to the appropriate titer in
solution 100 ml of antibody buffer. Label this solution “First
antibody solution.”
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