Which Western Blot Method is Right for Your Application?

For more than four decades, the Western blot method has been extensively used. Proteins are separated by electrophoresis according to their size through a gel, from where they can be moved to a membrane and then characterized using primary and then secondary antibodies.

It is essential that these antibodies form a band specific to the molecular weight on the membrane, which can be deciphered using the protein ladder run in tandem with the samples in the gel. As years passed, the detection techniques have been expanded and refined to enable more accurate and quantitative analysis of proteins; however, the universal technique has remained almost the same.

The technique selection will be based on the experiment and the availability of equipment in the lab. Gaining insights into the various options, the way they work, and the benefits they offer might render the identification of the protein much easier and contribute to the success of the experiment.

Radioactive Detection

Traditionally, antibodies for Western blot were labeled with radioactive labels. This radioactive detection technique started to lose its prominence with the development of other methods because of the risks posed by radiation exposure to researchers.

  • An X-ray film is exposed to the membrane, usually around 48 hours, to detect a band of protein.
  • With exposure to the radioactive tag, the film develops a dark band corresponding to the weight and amount of protein present.
. .
Advantages Sensitive
Disadvantages Health and safety risks, expensive, time-consuming
Top tip Avoid!

Enzymatic Detection

Colorimetric and Chemiluminescent

In this method, secondary antibodies that are linked to enzymes are used, thus requiring a substrate to function. These antibodies are either alkaline phosphatase (AP) or horseradish peroxidase (HRP) and can be used in either chemiluminescent or colorimetric detection techniques.

Spectrophotometry or densitometry (intensity of signal) can be used to quantitatively analyze the generated images. In order to carry out any quantitative analysis using densitometry methods, an appropriate loading control must be used in the experiment.


In colorimetric detections, the enzyme conjugated to the secondary antibody initiates a reaction with a substrate to form a colored precipitate.

  • Suitable substrate is added to membrane probed with antibodies
  • Insoluble precipitate builds up and stains the membrane, which can be seen by the naked eye
  • The development is stopped by washing the membrane, judged by the researcher’s own approximation

HRP in colorimetric detection is very economical but may lighten upon being exposed to light and can result in non-specific staining. On the contrary, AP in colorimetric detection yields a stable substrate that does not fade and can also enable the use of different substrates to develop different colors on the same membrane.

One main drawback of colorimetric detection is its sensitivity, which needs protein in the nanogram range to generate a signal. This is much more protein than it is required in other techniques, where a signal in the femtogram range can be detected.

. .
Advantage Fast, cheap, visual, no specialist equipment required
Disadvantages Not very sensitive, lots of protein required.
Top tips 1. Use for a quick and simple test for the presence or absence of a protein.
2. Use AP enzyme with different substrates to obtain more information from one membrane without stripping it.
3. Monitor the development of color closely to obtain the best image.


In chemiluminescent detections, the enzyme linked to the secondary antibody induces a reaction with a luminescent substrate that emits light as a by-product. With regards to colorimetric detection, the HRP and AP enzymes are used with suitable substrates.

  • The substrate is added to the membrane, with the substrate available in different strengths and stabilities that may be optimal for a particular experiment
  • The resulting light signal is detected by exposing it to X-ray film or charge-coupled device (CCD) imaging
  • Quantitative analysis is subsequently performed, which is largely dependent on the final image quality

The film detection technique is commonly considered the most sensitive; however, one has to work quickly while the enzymatic reaction remains stable on the membrane. CCD imaging is very effective since it is simple and automated; however, for extremely low expressed proteins, film exposure may be the safest option.

As there are no other means to differentiate bands of similar sizes and because of the possible cross-reactivity on the membrane, multiplexing different primary antibodies is complicated. The membrane can be stripped and re-probed but this can decrease the signal and can have an impact on the results.

. .
Advantages High sensitivity, best for low expressed proteins
Disadvantages Requires specialist equipment, multiplexing difficult.
Top tips 1. With film detection, try a few different exposure times to obtain the optimal image for quantification.
2. If you are struggling to get a band, try a different ECL substrate.
3. Leaving your membrane in wash buffer overnight then re-adding your substrate can reduce background of the image.

Fluorescent Detection

Fluorescent detection involves using secondary antibodies that are attached to particular fluorophores, hence there is no need for additional substrate; however, an exclusively designed digital imaging system is required.

  • With the help of this system, fluorophores are excited by LED, infrared, or visible light
  • Image is collected and quantified

The fluorophores generate a signal on their own that is a lot more stable compared to the enzymatic detection method, which lasts for months or even years. The fluorescent signal also has a considerably greater dynamic range when compared to chemiluminescence, thus there is better linearity in the detection limits. However, in comparison with enzymatic detection, fluorescence detection is usually considered less sensitive.

It is possible to use different fluorophore-tagged antibodies with different emission wavelengths on a single Western blot membrane, which implies that the experiment can be multiplexed without stripping and re-probing the membrane, hence there is no possibility of decreasing the signal.

. .
Advantages Stable, multiplexing straightforward
Disadvantages Less sensitive, requires specialist equipment.
Top tips 1. Take time to optimize the imaging system for the level of background noise, depending on the excitation window for the fluorophore.
2. Choose secondary antibodies with fluorophores emitting different wavelengths so you can multiplex and get the most out of a single experiment.
3. Protect your membrane from light to avoid the signal degrading, so you can re-image when necessary

Different detection methods for Western Blot including radioactive, enzymatic (colorimetric and chemiluminescent), and fluorescent analysis.Figure 1. Different detection methods for Western Blot including radioactive, enzymatic (colorimetric and chemiluminescent), and fluorescent analysis.

Final Remarks

Regardless of the detection method chosen, the resulting image will eventually rely on the whole design of the experiment, the sensitivity of the antibody, the use of appropriate controls, and also other optimization steps in the protocol.

The choice of detection method to be used may be solely made based on the laboratory or personal preference, or one may have the potential to trial a few options, and at times, scientific reasons can influence a particular choice.

  Sensitivity Multiplexing Enzyme Substrate Stability
Enzymatic: Colorimetric Low Possible AP advised Various Hours
Enzymatic: Chemiluminescent Highest Only with re-probing HRP advised Luminol based Hours
Fluorescent Low Easy None None Months - years

About Proteintech Group, IncProteintech Group, Inc

Proteintech: The Benchmark in Antibodies since 2001

Proteintech are a global biotech company and a renowned center of excellence for the manufacture and supply of quality antibodies, ELISA kits and proteins to the life science research community. With offices in the US (Chicago), UK (Manchester) and China (Wuhan) Proteintech are always available to support your research.

Part of Proteintechs early vision was to make all its own products, to the highest standards possible and to take complete responsibility for the quality. With an emphasis on developing antibodies from whole proteins, Proteintech provides researchers with unmatched reliability and reproducibility.

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Last updated: Feb 1, 2020 at 8:22 PM


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