ECL, DAB and NBT/BCIP colorimetric methods are very commonly used in biochemical experiments such as immunohistochemistry (IHC), immunocytochemistry (ICC) and western blot (WB).
Do you know what is the difference between them?
ECL——Enhanced chemiluminescence
ECL generally contains luminol, H2O2, luminous enhancers and other substances. Luminol is oxidized by HRP and H2O2 to produce fluorescence, and this process is enhanced by the luminescent enhancer.
The luminescent enhancer plays a key role in the system, the ideal luminescent enhancer should be (1) light-sensitive, (2) luminous stability, and (3) low background or no background.
DAB——3,3'-diaminobenzidine
DAB is a chromogenic substrate for peroxidases, which loses electrons in the presence of H2O2 and exhibits color changes and accumulation to form a tan insoluble product. The reaction is as follows:
HRP+H2O2+DAB→HRP+H2O+oxidized DAB↓
The method has the advantages of good specificity, stable performance and convenient operation. It is suitable for protein hybridization and immunochemistry.
BCIP——5-Bromo-4-chloro-3-indolyl phosphate
NBT/BCIP is one of the best substrate combinations for alkaline phosphatase (AP). Under the catalysis of AP, BCIP is hydrolyzed to produce a strong reactive product, which reacts with NBT to form insoluble dark blue to blue violet chemicals. The kit can be used for enzymatic coloration of IHC and WB experiments in alkaline phosphatase systems.
Table 1. Corresponding enzyme and reaction of ECL, NBT/BCIP and DAB
Classification
Enzyme
Coloration
Experiment
ECL
HRP
Fluorescence
WB Chemiluminescence Detection
DAB
HRP
tan
protein hybridization and immunochemistry, peroxidase method (enzyme linked immunosorbent assay, PAP method, etc.)
NBT/BCIP
AP
dark blue to blue-purple
IHC and WB in alkaline phosphatase system
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