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