Application of an enhanced luminol chemiluminescence reaction using 4-[4,5-di(2-pyridyl)-1H-imidazol-2-yl]phenylboronic acid to photographic detection of horseradish peroxidase on a membrane.
Photographic detection of horseradish peroxidase (HRP) on a membrane by the luminol-hydrogen peroxide-HRP chemiluminescence reaction using 4-[4,5-di(2-pyridyl)-1H-imidazol-2-yl]phenylboronic acid (DPPA) as an enhancer is described. The method is based on the long-lived chemiluminescence emission obtained by using DPPA. Under the optimum conditions, as little as 0.10 ng (ca. 2.3 fmol) and 0.20 ng (ca. 4.6 fmol) per spot of HRP on a membrane were detected as visible spots with exposure time of 60 and 10 min, respectively, by using an instant photographic film and a camera luminometer. The proposed method was highly sensitive and was successfully applied to the detection of HRP conjugates as an alternative to the colorimetric method using a chromogenic substrate in a commercially available assay kit of Western blotting.
Kuroda
N
N
School of Pharmaceutical Sciences, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852-8521, Japan. anal-kuro@cc.nagasaki-u.ac.jp
Murasaki
N
N
Wada
M
M
Nakashima
K
K
D015153:Blotting, Western; D001897:Boronic Acids / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000378:metabolism*; D008163:Luminescent Measurements; D008165:Luminol / Q000378:metabolism*; D010781:Photography
C428763:4-(4,5-di(2-pyridyl)-1H-imidazol-2-yl)phenylboronic acid; D001897:Boronic Acids; D008165:Luminol; D006735:Horseradish Peroxidase