Bio-Rad Immun-Blot® Opti-4CN™ Colorimetric Kits - Manual (Page 6)

for those steps. Antibody incubations should be at 0.1 ml
per cm
2 , e.g., 6 ml for a 60 cm 2 miniblot. Use dilute BAR
and streptavidin-HRP solutions at 85 µl per cm 2 of mem-
brane, e.g., 5 ml for a 60 cm 2 mini-blot. The prepared Opti-
4CN reagent is used at 0.25 ml per cm 2 of membrane.
Detergents. Tween-20 is essential in washing to elim-
inate overall background and non-specific hydrophobic
interactions. At 0.1%, Tween-20 will not disrupt binding
of primary antibodies to antigens or antigens to the mem-
brane, but will optimize detection sensitivity by eliminat-
ing non-specific interactions. Alternative detergents should
not be substituted.
2.2 Reagent Preparation
The following reagents should be made upon first
receiving the kit.
10x PBST (Phosphate buffered saline/1% Tween-20).
Pour the contents of the pouch into 950 ml ddH
2
O and
stir until dissolved. Add 10 ml Tween-20. Bring final vol-
ume to 1 liter with ddH
2
O. Store at room temperature.
1x PBST (Phosphate buffered saline/0.1% Tween-20).
Combine 100 ml 10x PBST and 900 ml ddH
2
O. Sterile
filter before use. Store at room temperature.
20% DMSO/PBST Wash. Combine 100 ml DMSO
and 400 ml 1x PBST. This is sufficient for 500 cm 2 of
membrane.
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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 pos-
itive signal without membrane background or non-spe-
cific reactions.
Secondary Antibody Conjugates. The protocols in
this manual were worked out using Bio-Rad Blotting Grade
secondary antibody conjugates diluted as described below.
Using an antibody conjugate at a higher concentration
may result in an overall increase in background without any
improvement in detection sensitivity. Secondary antibody
conjugates from other sources may be used, but the opti-
mal dilution may be different from that of Bio-Rad anti-
body conjugates.
Washes and Incubations. Continuous gentle agita-
tion should be used during all incubations and washes. For
best results, a rocking platform should be employed to
maintain a uniform exposure of the membrane surface to
the solution. Use the smallest possible container to hold
the membrane and solutions. When a range of washes is
specified, as in ‘Wash 2–4x’, best results are obtained by
doing the maximum number of washes. Acceptable results,
though potentially with more background, may be obtained
with the minimum number of washes. Blocking and wash-
ing steps should be done with 0.25 ml per cm
2 of mem-
brane, e.g., for a mini-blot of 60 cm 2 , use 15 ml of solution
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