Bio-Rad Immun-Blot® Opti-4CN™ Colorimetric Kits - Manual (Page 8)

a. Electrophoretic blotting. The antigens of interest are
electrophoretically transferred to the membrane from
a gel (i.e. SDS-PAGE, IEF or native gel) using the
Trans-Blot
® , Mini Trans-Blot ® , Trans-Blot Semi-Dry
cell or similar device.
b. Microfiltration blotting. The antigens of interest are
transferred by a vacuum device such as the Bio-Dot
®
or Bio-Dot SF onto the membrane. The membrane
should be removed from the apparatus for the block-
ing and all subsequent steps.
c. Dot blotting. Cut the membrane sheet to the appro-
priate size. Draw a grid on the membrane with a pen-
cil. Wet the dry membrane by slowly sliding the
membrane at a 45° angle into the PBST. (PVDF mem-
branes must first be wet in 100% methanol; consult
membrane instructions for complete information).
Remove the thoroughly wetted membrane from the
PBST and dry it on filter paper for approximately
5 minutes. Apply antigen sample to each grid square
using a syringe or pipet.
Note: Regardless of the method chosen for antigen appli-
cation, the membrane should be allowed to dry completely
before proceeding to the next step.
2. Wash. Wet the membrane in PBST and then wash the
membrane twice in PBST for 5 minutes each time.
These washes help to reduce spotted or blotchy back-
ground problems.
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Colorimetric Detection
14. Mix one part Opti-4CN diluent concentrate with
nine parts ddH
2
O. Prepare 0.25 ml per cm 2 of
membrane.
15. Add 0.2 ml of Opti-4CN substrate per 10 mls of
diluent (e.g., combine 1 ml Opti-4CN diluent con-
centrate with 9 ml ddH
2
O and 0.2 ml Opti-4CN
substrate). Mix well and pour onto membrane.
16. Incubate membrane with gentle agitation in the
substrate for up to 30 minutes or until the desired
level of sensitivity is attained.
17. Wash the membrane in ddH
2
O for 15 minutes.
18. Document or store membrane.
* GAx-HRP is Bio-Rad’s Goat Anti-Rabbit IgG-HRP (Catalog
number 170-6515) or Goat Anti-Mouse IgG-HRP (170-6516)
or Goat Anti-Human IgG-HRP (172-1050).
2.4 Detailed Protocol
Note: Before beginning, read through the entire procedure.
1. Antigen application. 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 produce a positive reaction if the pro-
cedure is successful.
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