ECL Western Blot Reagent

Chemiluminescence is the western blot detection method of choice in most protein laboratories, as it provides the greatest sensitivity and convenience for detection with film or digital imaging equipment.

Chemiluminescent substrates for horseradish peroxidase (HRP) are two-component systems consisting of a stable peroxide solution and an enhanced luminol solution. To make a working solution, the equal volumes of the two components are mixed together. When incubated with a blot on which HRP-conjugated antibodies (or other probes) are bound, a chemical reaction produces light that can be detected by film or sensitive camera (Figure 1).

ABP Biosciences offers two types of super sensitive chemiluminescent substrates for Western blot detection with horseradish peroxidase enzyme (HRP). The signal from ECL substrates can be captured and documented using X-ray film or a capable CCD camera imager.

 
Figure1. The principle of WB detection.

ECL Western Blot Reagent Selection Guide
 

	Immobilon™ Western (Millipore)	Super ECL	Super ECL Ultra
Sensitivity	picogram	low picogram to high femtogram	femtogram
Signal duration	<2 h	<8 h	<4 h
Primary Ab dilution	1:1,000-1:20,000	1:1,000-1:30,000	1:5,000-1:30,000
Secondary Ab dilution	1:20,000-1:200,000	1:50,000-1:200,000	1:100,000-1:300,000
Stability	1 year at 4℃	1 year at 4-25℃	1 year at 4-25℃

The comparison of sensitivity of Super ECL, Super ECL Ultra, and Millipore Immobilon™ Western blot substrate.

 
The comparison on exposure time.

 
The comparison on signal duration.

The comparison on antibody dilution.