Bio-Rad Immun-Blot® Opti-4CN™ Colorimetric Kits User Manual
Page 9
170-6515] or Goat Anti-Mouse IgG-HRP [170-6516]
or Goat Anti-Human IgG-HRP [172-1050]).
Secondary antibody HRP conjugates from other
sources may be used, but the optimal dilution will
have to be determined experimentally.
8.
Wash. Decant the antibody solution and add PBST to
the membrane. Wash for 5 minutes with gentle agita-
tion and pour off the wash solution. Repeat the wash
with fresh PBST.
Amplification (170-8238/39/40). All other kits,
skip to Detection.
9.
Amplification. Prepare 85 µl of 1x BAR solution per
cm
2
of membrane. Prepare the solution by combin-
ing 2 parts 2x Amplification diluent, 1 part 4x BAR
and 1 part ddH
2
O. Pour PBST off the membrane and
add the 1x BAR solution. Incubate for 10 minutes
with gentle agitation.
10. Wash. Pour off the BAR solution and wash the mem-
brane 2–4 times for 5 minutes each time with 20%
DMSO/PBST. Follow these washes with 1–2 washes
in PBST for 5 minutes each time.
11. Streptavidin-HRP. Prepare 85 µl of a 1:1,000 dilution
streptavidin-HRP per cm
2
of membrane. Dilute the
streptavidin-HRP in the same 1% BSA/PBST solu-
tion used to dilute the antibodies. Pour off the PBST
wash and add the diluted streptavidin-HRP solution.
Incubate for 30 minutes with gentle agitation.
13
3.
Blocking step. Immerse the membrane at a 45° angle
into the blocking solution. Gently agitate the solution
using a rocking platform and incubate for an hour or
more. Best results for amplification are attained with
a 3% solution of the blocker provided with the kits
(170-8238/39/40). For non-amplified applications,
5% blotto is an acceptable alternative.
4.
Wash. Decant the blocking solution and add PBST to
the membrane. Wash for 5 minutes. Repeat the wash
with fresh PBST.
5.
Primary antibody incubation. Decant the PBST and
prepare 0.1 ml of antibody solution per cm
2
of mem-
brane. Dilute the primary antibody in PBST with
1% (w/v) BSA. Incubate 1 to 2 hours with gentle
agitation. The optimum conditions of dilution and
incubation must be determined experimentally.
6.
Wash. Decant the antibody solution and add PBST to
the membrane. Wash for 5 minutes with gentle agita-
tion and pour off the wash solution. Repeat the wash
with fresh PBST.
7.
Secondary antibody incubation. Decant the PBST and
prepare 0.1 ml of the secondary antibody solution per
cm
2
of membrane. Dilute the secondary antibody
1:3,000–1:10,000 with PBST containing 1% (w/v)
BSA. Incubate for 30 minutes to 2 hours with gentle
agitation. As noted previously, this protocol was devel-
oped using Bio-Rad’s blotting grade secondary anti-
bodies (Goat Anti-Rabbit IgG-HRP [Catalog number
12
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