Protocol

Measuring Protein Concentration by Western Analysis Using Enhanced Chemiluminescence Detection

This protocol was adapted from "Rapid Dot-Blot Western Analysis Using PVDF Membranes and Enhanced Chemiluminescence Detection," contributed by Joint ProteomicS Laboratory (JPSL) of the Ludwig Institute for Cancer Research and the Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia, Appendix 2, in Purifying Proteins for Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2004.

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INTRODUCTION

In this protocol, samples containing the target protein are deposited onto a polyvinyldifluoride (PVDF) membrane using a vacuum manifold. The immobilized protein is exposed to an antibody specific for the target protein, followed by an antibody that reacts with species-specific determinants carried by the primary antibody and is conjugated to horseradish peroxidase (HRP). Antibody complexes captured by the immobilized target protein are detected by enhanced chemiluminescence (ECL); in the presence of H₂O₂, HRP converts luminol to an excited intermediate dianion that emits blue light (428 nm) on return to ground state (Isacsson and Wettermark 1974). Production of light . . . [Full Text of this Article]

MATERIALS

Reagents

Equipment

METHOD