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QuantaRed Chemifluorescent ELISA Substrate FAQ


Answers to frequently asked questions (FAQs) about QuantaRed Chemifluorescent ELISA Substrate


 

What is the QuantaRed Enhanced Chemifluorescent HRP Substrate?
QuantaRed Substrate is a proprietary formulation that uses chemical enhancers to increase fluorescent 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 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.

What is included in the QuantaRed Substrate Kit?
The kit includes QuantaRed ADHP Concentrate, Enhancer Solution, Stable Peroxide Solution and Stop Solution.

Can other wavelengths for excitation and emission be used to quantify HRP using this substrate?
Yes. Wavelengths at 530-575 nm for excitation and 585-630 nm for emission can be used for signal detection.

What type of instrumentation and filter sets are needed?
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.

Can the substrate be used colorimetrically?
Yes. The color produced by the reaction is dark magenta and can be measured at 570 +/-10 nm on a colorimetric plate reader.

What types of plates are compatible with the substrate?
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.

Can the signal be measured in a kinetic mode without stopping the reaction?
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.

Can a stop solution from another source be used?
No. The QuantaRed Stop Solution included in the kit must be used to effectively stop the reaction.

What antibody concentrations are recommended for use with QuantaRed Substrate?
The recommended concentrations of antibodies and HRP conjugate are listed in the table below:

Probe

Concentration (µg/ml)

Capture Antibody

1-10

Detection Antibody

0.05-1

HRP Conjugate

0.1-0.2

Can QuantaRed Substrate be used for Western blotting?
No. This is a soluble HRP substrate for ELISA and other plate-based applications.

 

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