Protocol

Western Analysis Using the Chemiluminescent Alkaline Phosphatase Substrate CSPD

This protocol was adapted from "Western Analysis Using the Chemiluminescent Alkaline Phosphatase Substrate CSPD," in Appendix 2 of Purifying Proteins for Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2004.

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

INTRODUCTION

Use of the chemiluminescence-producing alkaline phosphatase substrate 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1.1(3,7)]decan]-4-yl)phenyl phosphate (AMPPD, also known as adamantyl-1,2-dioxetane phosphate), or its dioxetane relatives (e.g., CSPD, a halogen-substituted derivative of AMPPD) provides a substantial increase in sensitivity over colorimetric substrates and radiochemical methods currently used for the detection of antigen-antibody complexes immobilized on nylon or PVDF membranes. With many antigen-antibody combinations, the method is capable of detecting as little as 1 pg of protein.

MATERIALS

Reagents

Blocking solution (e.g., Liquid Block, Amersham)

Prepare 5% (v/v) Liquid Block in PBS.

50X CAPS buffer

CAPS transfer buffer

CSPD assay buffer

0.1 mM CSPD/10% chemiluminescence enhancer

Primary antibody

Secondary antibody . . . [Full Text of this Article]

Equipment

METHOD