Which Western Blot Detection Method Should You Use?

Western blots can be detected with fluorescent, chemiluminescent, or colorimetric methods. Which 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. The, choose what works best for your research.

Fluorescent detection: Fluorescent detection uses secondary antibodies labeled with fluorescent dyes, rather than enzymes. No substrates are needed.

Enzymatic detection: Chemiluminescent and colorimetric methods use secondary antibodies labeled with enzyme reporters such as horseradish peroxidase (HRP). Signal-generating substrates are used.

Fluorescent detection uses NIR fluorescent dyes to generate a signal.
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 multiple protein targets without stripping and re-probing

Chemiluminescent detection
uses the horseradish peroxidase (HRP) enzyme and a luminescent substrate.
• 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 uses 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