Tips for Step 6: Block Endogenous Enzymes and Biotin

To avoid staining artifacts it is important:

  • To block endogenous peroxidases and phosphatases prior to using AP/horseradish peroxidase (HRP) antibody conjugates 

  • For blocking of endogenous peroxidase activity, use hydrogen peroxide solutions such as Bio-Rad’s ready-to-use Peroxide Blocking Reagent (BUF017B; 3% (v/v) hydrogen peroxide). However, for the detection of certain antigens, such as CD4, this percentage of hydrogen peroxide may be too harsh (Kim et al. 2016)

  • To block endogenous biotin when using avidin/biotin or streptavidin/biotin detection systems. For this specific purpose Bio-Rad offers a ready-to-use Avidin/Biotin Blocking Reagent (BUF016

  • To block endogenous AP activity supplement the substrate solution with levamisole (Ponder and Wilkinson 1981). According to Ponder and Wilkinson (1981), 20% acetic acid may also be used. However, this method may not be suitable for all antigens (Ponder and Wilkinson 1981)