Related Experiment Video
Updated: Oct 23, 2026
Western Blotting: Sample Preparation to Detection
Published on: October 14, 2010
A simple, two-color fluorescence detection method for membrane blotting analysis using alkaline phosphatase and
Y Kondoh1, S Fujita, N Kagiyama
1AISIN COSMOS R&D CO., LTD., Kariya, Aichi, Japan.
Abstract:
We have developed a one-step, two-color fluorescence detection method using simultaneously two fluorogenic substrates for both Southern and Western blots on nylon membranes. For this enzyme-mediated reporter system, a mixture of (i) 3-hydroxy-N-2'-biphenyl-2-naphthalenecarboxamide phosphate ester (HNPP), a substrate for alkaline phosphatase and (ii) N-(4-amino-5-methoxy-2-methylphenyl)benzamide (AMMB), a fluorogenic substrate for horseradish peroxidase was used. The reaction with these substrates produces blue (HNPP) and yellow (AMMB) fluorescent signals under ultraviolet light (302 nm). Therefore, this simple method allows the simultaneous visualization of two different targets on a single nylon membrane, e.g. nucleic acids or proteins.
Related Concept Videos
03:31Labeling DNA Probes
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
01:15Western Blotting
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.