Bio-Rad Mini-PROTEAN II Multiscreen Apparatus - Manual (Page 10)

4.4 Color Development of Enzyme Conjugates
Incubation of the blot with antibodies conjugated to enzymes, such as horseradish per-
oxidase or alkaline phosphatase, can be conducted either in the multiscreen apparatus or in a
separate vessel. If this step is carried out in the unit, wash the blot in the apparatus after the
second antibody incubation as outlined in Section 4.3.
1. Color development of enzyme conjugated antibodies should be performed in a separate
container to prevent permanent discoloration of the multiscreen apparatus. Remove the
membrane by loosening the four screws. Lift out the sample template and move the mem-
brane to a color development vessel.
2. Wash the membrane once with TBS for 5 minutes. After the color development solution
has been prepared, incubate the membrane in the solution. Gently agitate the solution
until development is complete. Remove the solution and rinse the membrane several
times in distilled water to stop the reaction. Air dry the blot on filter paper.
4.5 Detection with Colloidal Gold Conjugates
Incubation of the blot with colloidal gold conjugated antibodies, protein A, or protein G,
should be conducted in a separate vessel to prevent discoloration of the multiscreen apparatus.
1. Remove the blot from the multiscreen apparatus after washing to remove excess first
antibody. Place the membrane in a color development vessel.
2. Wash the membrane once with TBS for 5 minutes. Add the gold solution to the vessel until
the membrane is completely covered. Gently agitate the solution. Red bands identifying
antigen will appear on the membrane surface within 10–15 minutes at the sites of high-
est antigen concentrations. Allow the incubation to continue until the desired sensitivity
is achieved.
Section 5
Solutions for Immunoassay Applications
Tris Buffered Saline, 1 x TBS, 2 L
20 mM Tris-HCl, 500 mM NaCl, pH 7.5
Dissolve 4.84 g Tris, 58.48 g NaCl in ~1.5 L distilled, deionized H
2
O. Adjust to pH 7.5
with HCl. Adjust the volume to 2 L with dd H
2
O.
Note: Bio-Rad's Premixed Tris-Buffered Saline (catalog number 170-6430) eliminates
weighing of buffer components. One bottle produces 1 L of 10 x TBS.
Tween-20 Wash Solution, 1X TTBS, 1 L
20 mM Tris-HCl, 500 mM NaCl, 0.05% Tween-20, pH 7.5
Add 0.5 ml Tween-20 to 1 L of TBS.
Blocking Solution, 100 ml
Both of the following blocking solutions can be used with nitrocellulose. The solutions
containing BLOTTO should be used with Zeta-Probe membrane. Incubate nitrocellulose
blots for 1 hour at room temperature. Zeta-Probe membrane should be blocked for 2 hours
at room temperature.
3% Gelatin - TBS
Add 3.0 g gelatin to 100 ml TBS. Heat to 50 °C, stirring until dissolved. A microwave
oven will quickly solubilize the gelatin, but do not heat above 65 °C.
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