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High sensitivity can, however, be disadvantageous in samples that present high background and the end-user must still examine how the affinity of the protein, primary antibody, secondary antibody, and reporter enzyme substrate will vary in performance from one sample to another and make appropriate optimizations.Ĭhemiluminescent-based methods offer much higher sensitivity relative to colorimetricbased methods that detect proteins within the 100pg-500pg range or fluorescentbased methods which are limited to the nanogram (10-9) range. Given the short lived nature of chemiluminescent signals, enhancer chemiluminescent substrates (ECL) have been developed which enable increased signal duration, intensity, and sensitivity.
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HRP-substrate reactions can detect down to 1 to 3 pg and AP-substrate reactions can detect as little as 10 pg of protein. One advantage of chemiluminescent Western blot systems is the sensitivity. Both methods are compatible on nitrocellulose or PVDF membrane blots. Once an end-user has optimized the correct concentration of enzyme-conjugated antibody, a transient chemiluminescent signal is produced that decays quickly upon exhaustion of substrate. Chemiluminescent systems are easily adapted to traditional Western blotting protocols due to the use of enzyme-conjugated antibodies to activate the light signal. These light signals are then captured on X-ray film or charge-coupled device (CCD) cameras as the light signal decays to the ground state. AP dephosphorylates 1, 2-dioxetane-based substrates to yield a dioxetane phenolate anion that emits a light signal at 466nm. When utilizing AP, 1, 2-dioxetane-based reagents are the adjacent AP substrates. HRP in a peroxide buffer oxidizes luminol to its excited state product, 3-aminophthalate, which emits a light signal at 425nm. If utilizing HRP, luminol-based reagents are selected. The choice of substrate for chemiluminescent Western blotting is determined by which reported enzyme is chosen. The two commonly used enzyme reporters are HRP and AP. It contains a complete reagent set, with optimized ready-to-use or ready-to-dilute reagents.Ĭhemiluminescent detection systems take advantage of energy released in reporter enzyme-substrate reactions in the form of light to be the recorded signal. The kit can be used to identify primary mouse antibodies on a Western blot using immunodetection. The new Chromogenic Western Blot Kit allows for quick, visual reading of Western blots in unpurified cell extract. Using the size and color intensity of the protein band, a semi-quantitative estimation of protein can be derived. When the enzyme substrate is added, either a colored precipitate (colorimetric detection) or a chemiluminescent or fluorescent product is formed and the light signal is captured. Immobilized membrane-bound proteins are generally detected using secondary antibodies that are conjugated to fluorescent molecules (fluorophores) or an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP). Due to its flexibility, Western blotting can be used for a wide range of applications within molecular biology and biochemistry, including detection of post-translational modifications and verification of protein cloning.