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View additional product information for QuantaRed™ Enhanced Chemifluorescent HRP Substrate Kit - FAQs (15159)
10 product FAQs found
No. This is a soluble HRP substrate for ELISA and other plate-based applications.
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The recommended concentrations of antibodies and HRP conjugate are listed below:
Capture Antibody: 1-10 µg/mL
Detection Antibody: 0.05-1 µg/mL
HRP Conjugate: 0.1-0.2 µg/mL
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No. The QuantaRed Stop Solution included in the kit must be used to effectively stop the reaction.
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To measure in a kinetic mode, simply omit the Stop Solution. At high HRP concentrations and prolonged reaction times, the fluorescent product may self-quench, which is observed as continuous color development without a change in fluorescent intensity. Optimization of the detection antibody or HRP conjugate is required to determine the linear range of a particular assay system.
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Black opaque plates typically produce the best results. White opaque plates also can be used, but might produce higher backgrounds. A variety of other plate types, such as gray or opaque plates with transparent bottoms are also useful with this substrate. Use clear plates if the absorbance of the colored product will be measure instead of the fluorescence.
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Yes. The color produced by the reaction is dark magenta and can be measured at 570 +/-10 nm on a colorimetric plate reader.
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The QuantaRed Signal can be measured using standard fluorometers equipped with Cy3 Dye filter sets. Quantitation does not require filters that precisely match the excitation/emission maxima; however, a non-overlapping filter set with a bandpass that includes the excitation/emission spectra is required.
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Yes. Wavelengths at 530-575 nm for excitation and 585-630 nm for emission can be used for signal detection.
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The kit includes QuantaRed ADHP Concentrate, Enhancer Solution, Stable Peroxide Solution and Stop Solution.
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QuantaRed Enhanced Chemifluorescent HRP Substrate is a proprietary formulation that uses chemical enhancers to increase fluorescence yield and sensitivity of 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) chemifluorescence in plate-based (e.g., ELISA) assays. ADHP is a non-fluorescent compound that reacts with horseradish peroxidase (HRP) in the presence of peroxide to produce resorufin, a soluble, highly fluorescent compound with excitation/emission maxima at 570/585 nm. Unlike chemiluminescent substrates, the QuantaRed Enhanced Chemifluorescent HRP Substrate reaction can be stopped with the fluorescent signal remaining stable for several hours. The long wavelength emission spectrum of resorufin helps minimize interference from lower wavelength autofluorescence (blue and green) that exists in many biological samples.
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