title: A cost effective non-commercial ECL-solution for Western blot detections yielding strong signals and low background.
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.
authors.affiliation: 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
authors.forename: Claude
authors.initials: C
authors.lastname: Haan
authors.forename: Iris
authors.initials: I
authors.lastname: Behrmann
mesh_terms: D015153:Blotting, Western / Q000379:methods*; D001896:Boron Compounds / Q000737:chemistry; D045744:Cell Line, Tumor; D003373:Coumaric Acids / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007462:Iodobenzenes / Q000737:chemistry; D053613:Janus Kinase 1 / Q000032:analysis / Q000276:immunology; D049449:Luminescence; D008165:Luminol / Q000737:chemistry; D011422:Propionates; D012996:Solutions / Q000737:chemistry
chemical_list: D001896:Boron Compounds; D003373:Coumaric Acids; D007462:Iodobenzenes; D011422:Propionates; D012996:Solutions; C080454:4-iodophenylboronic acid; C050181:4-iodophenol; D008165:Luminol; D006861:Hydrogen Peroxide; D053613:Janus Kinase 1; C495469:p-coumaric acid