Quantification of Protein in Brain Tissue by Western Immunoblot Analysis - Drugs of Abuse: Neurological Reviews and Protocols - page 262

Fig. 2. Development of the PVDF membrane with the antibody sandwich and
luminol. (A) Ten samples were loaded onto a gel, and proteins were transferred to a
PVDF membrane. Antigen for the primary antibody is present in all even lanes, but
not odd lanes. The primary antibody binds to the antigen, which is immobilized on
the membrane. The secondary antibody has the primary antibody as antigen (1) . The
secondary antibody is linked to horseradish peroxidase. (B) Sandwich of the antigen,
primary antibody, and secondary antibody linked to horseradish peroxidase. After
addition of hydrogen peroxide and luminol (in the substrate bottles provided by the
manufacturer), horseradish peroxidase proceeds to metabolize hydrogen peroxide, a
reaction that causes the oxidation/ excitation of luminol. When luminol converts to
the ground state, light is emitted.

with gentle shaking. ( Note : Dilution of secondary antibody is usually between
1 30,000.) Secondary antibody is directed against IgG from the

host species of the primary antibody, and conjugated to horseradish peroxidase.
For more information on antibody reactions, see ref. 1 . Note: If the background is
too high, primary and secondary antibody can be diluted in blocking solution.

10,000 and 1

6. Wash four times for 7-10 min in PBST at room temperature with gentle shaking.

3.5. Development of Antibodies
Different reagents are available from various companies which are provided
with the appropriate protocols. The following protocol is for Luminol Reagent