Update profile
    Change password
    Sign out

QuantaRed Enhanced Chemifluorescent HRP Substrate


Harness the sensitivity of fluorescence for ELISA-based applications.

QuantaRed Enhanced Chemifluorescent HRP Substrate

The Thermo Scientific QuantaRed Enhanced Chemifluorescent HRP Substrate is the most sensitive fluorescent ELISA substrate, providing sensitivity comparable to enhanced chemiluminescence.

QuantaRed HRP Substrate enables high-yield and sensitive fluorescent and colorimetric detection of peroxidase activity in the ELISA format. QuantaRed Substrate uses a proprietary technology to increase fluorescent yield and sensitivity of the well-documented ADHP (10-Acetyl-3,7-dihydroxyphenoxazine) chemifluorescence reaction. ADHP is a non-fluorescent compound that reacts with horseradish peroxidase (HRP) to produce resorufin, a soluble, highly fluorescent reaction product with excitation/emission maxima of ~570/585nm. The long wavelength emission of the resorufin minimizes interference from the low wavelength (blue and green) autofluorescence, making it an ideal substrate for quantitating target molecules in biological samples. In addition, the reaction product of QuantaRed Substrate is colorimetric as well as being fluorescent and can be read spectrophotmetrically at ~570nm.

Highlights:

  • Sensitive – low picogram to femtogram detection
  • Red-Shifted - minimal autofluorescence interference from biological samples
  • Convenient – compatible with existing filter sets, no need to purchase special filter sets
  • Complete – mix and use reagents, no buffers to prepare or reagents to dissolve
  • Flexible – use Stop Solution to effectively stop the reaction resulting in stable signal for at least 4 hours or more.
  • Versatile – produces a strong colorimetric signal and can be read fluorescently or colorimetrically at 570nm

Product Details:

Specially formulated, QuantaRed Substrate incorporates chemical enhancers for maximum sensitivity and superior low end linearity. With high quantum yield and large extinction coefficient, QuantaRed Enhanced HRP Substrate has sensitivity comparable to enhanced chemiluminescence and is more sensitive than the Amplex Red substrates for the detection of HRP in ELISA applications.

QuantaRed Spectrum Data: Excitation=572 nm, Emission=588 nm QuantaRed Fluorescent ELISA Substrate enables a red-shifted spectrum. Normalized absorption and fluorescence emission spectra of resorufin, the reaction product of QuantaRed Enhanced Chemifluorescent HRP Substrate.

A.
QuantaRed Enhanced Chemifluorescent HRP Substrate enables low picogram target detection. Wells of an opaque black 96-well microplate were coated with 1µg/mL Mouse anti-Human IL-17 (from Part No. ESS0030) and blocked with ELISA Blocker Blocking Buffer. A standard curve (plotted) and Phytohemagglutinin (PHA) stimulated and unstimulated peripheral blood mononuclear cells (PBMCs) were tested. After incubation for 1 hour, the wells were washed and then biotinylated mouse anti-Human IL-17 was added at a concentration of 0.1µg/mL for 1 hour. After washing again, a 1:2000 dilution of the Ultra Streptavidin-HRP included in the kit was added to the wells and incubated for 30 minutes. After a final wash cycle, 100µL of prepared QuantaRed Substrate working solution was added for a 5-minute incubation before adding the QuantaRed Stop Solution. The fluorescent signal was measured using a Tecan Safire Fluorometer with excitation/ emission at 560/590nm. The detection range for the recombinant human IL-17 was 8-5000pg/mL (Panel A). The detection limit for the assay was 1.6pg/mL (160fg, Panel B). Through linear regression the PHA- stimulated PBMCs were calculated to contain 16.5pg/mL Human IL-17 while the unstimulated samples contained less than 1.6 pg/mL.
B.

A.
QuantaRed Substrate outperforms Amplex Red and Amplex Ultra Red HRP Substrates. QuantaRed Enhanced Chemifluorescent Substrate was compared to ADHP-based chemifluorescent substrates from other suppliers using the procedure outlined in Thermo Scientific Human IFNγ ELISA Kit (Part No. EHIFNG). Both colorimetric (Panel A) and fluorescent (Panel B) signals were measured. The detection range of recombinant Human IFNγ using colorimetric detection of absorbance at 572nm was 4-2500pg/mL for the QuantaRed Enhanced Chemifluorescent HRP Substrate. Amplex Red was able to detect only 100-2500pg/mL and Amplex UltraRed only 20-2500pg/mL. The fluorescent detection range for the QuantaRed Substrate was 0.16-2500pg/mL. Amplex Red and Amplex UltraRed both detected in the range of 4-2500pg/mL.
B.

Related Resources:

QuantaRed Chemifluorescent Substrate FAQ
Assay development and ELISA technical guide
ELISA substrate selection guide

Related Products:

QuantaBlu Fluorogenic Peroxidase Substrate


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
15159 QuantaRed Enhanced Chemifluorescent HRP Substrate Kit
Formulation: Four-component kit
Sufficient For: 960 wells (10 x 96-well plates) at 100&muL working solution per well

Kit Contents:
QuantaRed ADHP Concentrate, 1mL
QuantaRed Enhancer Solution, 50mL
QuantaRed Stable Peroxide Solution, 50mL
QuantaRed Stop Solution, 10mL
110mL kit Product Instructions for product #15159 QuantaRed Enhanced Chemifluorescent HRP Substrate Kit MSDS for product #15159 QuantaRed Enhanced Chemifluorescent HRP Substrate Kit Certificate of Analysis for product #15159 QuantaRed Enhanced Chemifluorescent HRP Substrate Kit $312.00

Add to cart

Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

Recommend this page

Follow Us
Email Sign-up  Email Announcements

You may also need
21059 - Restore Western Blot Stripping Buffer

46430 - Restore PLUS Western Blot Stripping Buffer

28904 - Trifluoroacetic Acid (TFA), Sequencing grade

28908 - 16% Formaldehyde (w/v), Methanol-free

24590 - GelCode Blue Stain Reagent

 



Antibodies  |   Molecular Biology   |   Cell Biology   |  Thermo Scientific  |   * Trademarks  |   Privacy Statement
© 2014 Thermo Fisher Scientific Inc.   |   3747 N Meridian Rd, Rockford, IL USA 61101   |   1-800-874-3723  or  815-968-0747