Western Blot Detection Methods: Fluorescence, Chemiluminescence, and Colorimetric?

Western blots can be detected with fluorescent, chemiluminescent, or colorimetric methods. What method do you use?

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