Bio-Rad Immun-Star™ AP Chemiluminescence Kits - Manual (Page 11)

Blotting Grade Conjugates. The conjugates supplied by
Bio-Rad should be used in the concentrations indicated in
Section 2.3. Using a conjugate at higher concentrations may
result in an overall increase in background without any increase
in detection sensitivity.
Washes and Incubations. Continuous gentle agitation
should be used during all reactions. For best results, an orbital
shaker should be employed to maintain a uniform exposure of
the membrane to the solution.
Detergents. Tween 20 is essential in washing to eliminate
overall background and nonspecific hydrophobic reactions. At
0.05%–0.1%, Tween 20 will not disrupt binding of primary
antibodies to antigens or antigens to the membrane, but will
optimize detection sensitivity by eliminating nonspecific
reactions. Increased concentrations of Tween 20 (up to 0.3%)
can be used if background problems persist. Alternative
detergents should not be substituted. The wash between the
blocking step and incubation with the first antibody is
essential and should not be altered.
Molecular Weight Standards. Biotinylated SDS-PAGE
standards and Precision Plus Protein
™ unstained standards are
recommended for molecular weight determinations with the
Immun-Star assays. The Biotinylated SDS-PAGE standards are
detected by binding avidin-AP or streptavidin-AP to the biotinyl-
ated proteins, which will produce a chemiluminescent signal
upon reaction with the substrate. The Precision Plus Protein
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