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Methods commonly used to quench endogenous peroxidases, phosphatases, and biotin interferences
| If Using ▲ ▼ | Block ▲ ▼ | Tissue Types ▲ ▼ | Blocking Method ▲ ▼ |
| Biotinylated antibodies | Biotin | Liver, kidney, heart, brain, lung, mammary gland, and adipose tissue | To block endogenous biotin, first, incubate the tissue sample with streptavidin. Since streptavidin is tetrameric and can bind up to four biotin molecules, a subsequent incubation step with free biotin is required to block the remaining unoccupied biotin-binding sites. |
| HRP-labeled antibodies | Peroxidases | Liver, kidney, and vascular areas with red blood cells | Tissues can be tested for endogenous HRP by incubating with ABTS or TMB prior to primary antibody incubation. If a blue-green or blue color is observed, respectively, HRP is present, and blocking is required. Incubate tissue sections in 3% hydrogen peroxide for 10 minutes to quench endogenous peroxidase activity. Then wash three times with PBS. For delicate samples, where 3% hydrogen peroxide may damage the section or alter epitope accessibility, try a lower concentration of 0.3%. |
| AP-labeled antibodies | Phosphatases | Intestine, kidney, osteoblasts, lymphoid tissues, and placenta | Tissues can be tested for endogenous AP by incubating with substrate NBT/BCIP prior to primary antibody incubation; AP is present if a blue color is observed, and blocking is required. Quench endogenous phosphatase activity using 1 mM Levamisole; this is added with the chromogenic substrate. For intestinal AP, Levamisole has no effect. Block intestinal AP with 1% acetic acid before primary antibody incubation. |
References
This online tool may be cited as follows
MLA | "Quest Database™ Methods commonly used to quench endogenous peroxidases, phosphatases, and biotin interferences." AAT Bioquest, Inc., 20 Aug. 2025, https://www.aatbio.com/data-sets/methods-commonly-used-to-quench-endogenous-peroxidases-phosphatases-and-biotin-interferences. | |
APA | AAT Bioquest, Inc. (2025, August 20). Quest Database™ Methods commonly used to quench endogenous peroxidases, phosphatases, and biotin interferences. AAT Bioquest. https://www.aatbio.com/data-sets/methods-commonly-used-to-quench-endogenous-peroxidases-phosphatases-and-biotin-interferences. | |
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