Bio-Rad Immun-Star™ AP Chemiluminescence Kits - Manual (Page 10)

Water Purity. Use only deionized distilled water to prepare
all solutions. In addition, care should be taken to prevent
alkaline phosphatase contamination of assay solutions.
Ideally, distilled deionized water (ddH
2
O) should be autoclaved
or sterile-filtered prior to use in buffers and solutions.
Membrane Selection. The Immun-Star assay is specially
designed for use with nitrocellulose and PVDF membranes.
This kit is not compatible with nylon membrane. The use of
Immun-Star enhancer is required when performing blots on
nitrocellulose membrane. Longer exposure times will be
necessary if the enhancer is not used, with resulting interfer-
ence from background development. If proteins are
transferred to PVDF membrane, use of the enhancer is not
necessary. All other steps in the assay procedure remain the
same.
Primary Antibody. Generally, when serum or tissue culture
supernatants are the source of primary antibody, a
1:100–1:1,000 dilution of the primary antibody in buffer is
used for detection of antigens on the membrane surface. For
chromatographically purified monospecific antibodies, a
1:500–1:10,000 dilution in buffer is used for antigen detection.
A 1:1,000–1:100,000 dilution is used when ascites fluid is the
source of antibody. Optimal dilution factors must be deter-
mined experimentally. The optimal antibody concentration is
usually considered to be the greatest dilution of antibody
reagent that still results in a strong positive signal without
significant membrane background or nonspecific reactions.
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