Visualization under ultraviolet light enhances 100-fold the sensitivity of peroxidase-stained blots.

Analytical Biochemistry
A Domingo, R Marco

Abstract

As described in this article, visualization and/or photography under uv light of 4-chloro-1-naphthol-developed, peroxidase-marked immunoblots allows an increase in sensitivity of more than 100 times over the apparent staining results observable under normal visible white light. This increase in sensitivity can be obtained with the minimal additional requirement of an uv lamp, with the actual chloronaphthol staining procedure remaining unaltered and thereby allowing the monitoring of specific reactions with much smaller quantities of antigen or antibodies. Substantial shortening of the procedure is another advantage, making it possible to complete in 20 min or even less a procedure usually requiring 3 to 6 h. The phenomenon depends on the uv absorption and the fluorescence quenching properties of the products of the peroxidase reaction. The absorption spectra of the membranes with or without peroxidase products indicate that an intermediate in the peroxidase reaction is responsible for the absorption under uv light. This intermediate accumulates under conditions where the final product absorbing in the visible light has not begun to be produced, thus explaining the large increase in sensitivity. The behaviors of three types of m...Continue Reading

References

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Citations

Jun 24, 1992·Journal of Immunological Methods·S Avrameas
Nov 15, 2002·Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology·Takahiko AokiRyuji Ueno
May 1, 1997·Brain Research. Brain Research Protocols·F NajemeJ Bonnet
Aug 15, 1991·Proceedings of the National Academy of Sciences of the United States of America·K SaitoI Murakoshi
Oct 26, 2010·Analytical Biochemistry·Thorsten KucziusJan P M Langeveld
Jul 20, 2016·Applied Microbiology and Biotechnology·Florian GuggenbichlerChristoph Pöhlmann

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