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. Chemiluminescence is likely the most common. Other means of detection include fluorescence, near-infrared fluorescence, colorimetric, and radioactive.
Explore: Western Blot Imaging Systems
What is chemiluminescent detection?
In Western blotting, 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
What are the advantages of using fluorescent Western blot vs. chemiluminescent Western blot?
1. Fluorescent Western blotting allows multiplexing. By using different fluorescent dyes with non-overlapping excitation and emission spectra, multiple proteins can be assayed on one blot without needing to strip and re-probe the blot.
2. Fluorescent detection is more quantitative than chemiluminescent detection. Chemiluminescent detection relies on an enzyme (HRP or AP) bound to the antibody, and the activity of the enzyme can change depending on conditions and as the amount of substrate changes. Fluorescent detection relies on the emission of light from a fluorescent probe bound to the antibody. The fluorescence intensity will only depend on the number of fluorescent molecules present in a given spot.
Continue reading: Alternative Total Protein Stains for Fluorescent Western Blots
Is HRP a chemiluminescent substrate?
No! Even though it is an important component of chemiluminescent detection, HRP stands for horseradish peroxidase, an enzyme isolated from the roots of the horseradish plant. HRP catalyzes the oxidation of substrates, transferring electrons from the substrate to peroxide. In chemiluminescent Western blot detection, HRP is conjugated to an antibody and the location of the antibody on a blot is detected by incubating the blot with a substrate that will produce light after it is oxidized by the HRP enzyme.
Explore: HRP Stripping Buffer
What is a chemiluminescence substrate?
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.
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