Which Western Blot Detection Method Should You Use?

Western blots can be detected with fluorescent, chemiluminescent, or colorimetric methods. Which Western blot detection method should you choose?

Find out how the three common Western blot detection methods compare to each other in terms of time, sensitivity, and other important factors. Then, choose what works best for your research.

Fluorescent detection

Fluorescent detection uses NIR fluorescent dyes to generate a signal. This method uses secondary antibodies labeled with fluorescent dyes, rather than enzymes. No substrates are needed.

Secondary antibodies are labeled with dyes such as IRDye 800CW or IRDye 680RD
Digital imaging reveals target protein signals with high sensitivity
Quantitative (signal is proportional to the amount of target protein present)
Stable fluorescent signals are stable
Multiplex detection of several protein targets without stripping and re-probing

Chemiluminescent detection

Chemiluminescent detection uses secondary antibodies labeled with enzyme reporters such as horseradish peroxidase (HRP) and a luminescent substrate. Signal-generating substrates are used.

Enzymatic reaction produces light that is detected by film exposure, or digital imaging with CCD camera
Multiple exposures typically required to capture optimal signals and avoid signal saturation
Very sensitive
Cannot be multiplexed
May not be quantitative

Colorimetric detection

Colorimetric detection uses secondary antibodies labeled with the alkaline phosphatase enzyme.

Enzyme converts a soluble chromogenic substrate to a colored, insoluble product that precipitates onto the membrane and produces colored bands
Development of the blot is stopped by washing away the soluble substrate
Simple and cost-effective
Limited sensitivity

Comparison of Chemiluminescence and Infrared Fluorescence Detection for Western Blotting

	Chemiluminescence	IR Fluorescence
Sensitivity	+++	+++
Linear Dynamic Range	10-50 fold	>4000 fold
Multiplex Detection	No	Yes
Signal Stability	Hours	Months - Years
Enzyme Conjugate	HRP	---
Substrate	Luminol-based	None Needed
Detection/Documentation	Film Exposure/Digital Imaging	Digital Imaging
Membrane Compatibility	Nitrocellulose or PVDF	Nitrocellulose or PVDF

Learn more about why near-infrared fluorescence detection is the best choice for quantitative, replicable Western blots.