Cite as: Cold Spring Harb. Protoc.; 2006; doi:10.1101/pdb.prot4335

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Detection of Alkaline Phosphatase-Labeled Reagents

Ed Harlow and David Lane

This protocol was adapted from "Staining Tissues," Chapter 6, in Using Antibodies by Ed Harlow and David Lane. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1999.

The first 100 words of the full text of this article appear below.


INTRODUCTION

Alkaline phosphatase-linked secondary reagents are best used for low-resolution studies that are aimed at the rapid detection of the presence or localization of the antigen. The major advantages of using this detection method are the sensitivity and the ability to observe the results with just a light microscope. Extra sensitivity can be found by observing the precipitated enzyme products by interference reflection microscopy. Bromochloroindoyl phosphate/nitro blue tetrazolium (BCIP/NBT) is the most commonly used of the chromogenic substrates for alkaline phosphatase. The BCIP/NBT substrate generates an intense black-purple precipitate at the site of enzyme binding. The reaction proceeds at a steady . . . [Full Text of this Article]


MATERIALS

Reagents

Equipment


METHOD


TROUBLESHOOTING


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