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Your common Western blotting questions, answered.

Western blotting is a widely used analytical technique that can identify one or more specific proteins in a complex mixture of proteins. It is a powerful tool that provides information about the presence, size, and under the right conditions, even the amount of a protein. Though commonly used and often routine in many labs, Western blotting can be source of frustration when it doesn’t work. It involves several steps, each of which needs to be optimized to achieve the best results. The key to the best Westerns is understanding the process. We’re here to help with some answers to your most commonly asked Western blotting questions.

What are some of the detection methods used in Western blotting?

Several options are available to detect Western blots, with chemiluminescence is likely the most common. Other means of detection include fluorescence, near-infrared fluorescence, colorimetric, and radioactive.

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What is chemiluminescent detection?

Chemiluminescent detection is a method of detecting the location of antibodies bound to a Western blot. Chemiluminescent detection relies on an enzyme, either horseradish peroxidase or alkaline phosphatase, bound to an antibody. The enzyme converts a substrate to a product that emits light (chemiluminescence). The light emitted can be detected on X-ray film or by CCD camera.

What's more sensitive: chemiluminescence or fluorescence?

In general, fluorescent detection can detect picograms of protein while chemiluminescence can detect protein in the femtogram range.

However, sensitivity of detection depends on many things. The ability to detect small amounts of target protein requires a high-quality primary antibody with high affinity and specificity for the target protein. In addition, with CCD cameras, very long exposures are possible to maximize the chance of detecting a low-abundance band but this requires minimizing background “noise” on the Western blot. In addition, different fluorophores have different quantum yields, and some HRP substrates are engineered to increase sensitivity, so the sensitivity of fluorescent detection depends on the specific fluorophore used, and the sensitivity of chemiluminescent detection depends on the substrate used.

Radiance Q chemiluminescent substrate is designed to produce a strong, long-lasting signal for large linear dynamic range and quantitative data

Continue readingBeginning Chemiluminescent Western Blotting
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What are the advantages of using fluorescent Western blot vs. chemiluminescent Western blot?

Diagram of how fluorescent Western blotting can detect two proteins in two spectrally different channels.
Multiplex detection is possible by using two or more fluorescent dyes and an instrument that can excite and detect the light from each dye.

Is HRP a chemiluminescent substrate?

Diagram illustrating the principles of chemiluminescent Western blotting
The principle of chemiluminescent Western blotting

What is a chemiluminescence substrate?

luminol chemical formula
Luminol chemical formula

Chemiluminescent substrates produce light in the presence of HRP and hydrogen peroxide. An example of a chemiluminescent substrate is luminol. Luminol is oxidized to 3-aminophthalate which emits light (chemiluminescence) that can be detected on X-ray film or by a CCD camera.

Additional Western Blotting Resources

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New to Western blotting? Need to troubleshoot your Western blot?​ Want to brush up on Western blotting best practices? Claim your free Western Blotting eBook!

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