Bio-Rad Immun-Star™ AP Chemiluminescence Kits - Manual (Page 12)

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unstained standards are detected by incubating with
StrepTactin-AP. Because the avidin-AP (or streptavidin-AP) and
StrepTactin-AP can be directly added to the second antibody
solution, no extra steps are needed to detect the biotinylated or
Precision Plus Protein unstained standards. The standards
should be diluted 1:50 in electrophoresis sample buffer (note:
this is a higher dilution than recommended in the biotinylated
standards manual). Load 10 µl per lane for mini gels and 15 µl
per lane for full size gels.
Prestained SDS-PAGE and Kaleidoscope prestained
standards can also be used for assessing the transfer
efficiency of samples.
Total Protein Detection. In the identification of specific
antigens, total protein staining is required to correlate the
signal detected to a complex protein mixture. Any of the
common total protein staining methods will work to give a
profile of the blot, including colloidal gold, Amido Black, and
Ponceau S. If you want to generate a chemiluminescent total
protein blot, there are Biotin Blot
™ systems that allow
visualization by chemiluminescent signal. The chemilumines-
cent detection method uses NHS-biotin to biotinylate all the
proteins on the membrane. The biotinylated proteins are
detected with avidin-AP or streptavidin-AP and visualized with
the chemiluminescent substrate (see Appendix 1 for details).