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J Immunol Methods. 2007 Jan 10;318(1-2):11-9. Epub 2006 Nov 2.

A cost effective non-commercial ECL-solution for Western blot detections yielding strong signals and low background.

Author information

1
Laboratoire de Biologie et Physiologie Intégrée, Fac. Science Techno. and Com., Université du Luxembourg, 162a, Avenue de la Faïencerie, L-1511 Luxembourg, Luxembourg. claude.haan@uni.lu

Abstract

We compared several alternative ECL solutions for Western blot detection of endogenous proteins in whole cell lysates using inexpensive, commercially available reagents. Starting from an existing protocol based on p-coumaric acid (pCA) as enhancer, we found that the ECL solution containing 4-iodophenylboronic acid (4IPBA) generated strong specific signals and low background chemiluminescence. We optimised the luminol, 4IPBA and hydrogenperoxide concentrations of this 4IPBA-ECL solution. The optimised 4IPBA-ECL solution (100 mM Tris/HCl pH 8.8, 1.25 mM luminol, 2 mM 4IPBA, 5.3 mM hydrogenperoxide) shows a greatly increased signal intensity compared to the initial pCA-ECL protocol and to some commercially available ECL solutions. In addition, the optimised 4IPBA-ECL solution also generates much lower background chemiluminescence than other non-commercial ECL solutions using p-coumaric acid or 4-iodophenol as enhancers. The 4IPBA-ECL solution was stable when stored but had the lowest background when prepared freshly from stock solutions. Thus, we present an optimised protocol for a well-performing inexpensive ECL solution which is an alternative to expensive commercial ECL solutions and which achieves a better signal and lower background than the commercial solutions tested.

PMID:
17141265
DOI:
10.1016/j.jim.2006.07.027
[Indexed for MEDLINE]

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