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Horseradish Peroxidase (HRP) and Poly-HRP

Horseradish peroxidase (HRP) is a popular enzyme used extensively in molecular biology to detect immune complexes and biological targets such as proteins, carbohydrates, and nucleic acids. HRP is typically conjugated to antibodies, streptavidin, or other proteins and functions as a reporter system for probe-based immunoassay applications, including ICC, IHC, Western blotting, in situ hybridization (ISH), and ELISAs.

AAT Bioquest offers a complete portfolio of HRP labeling tools and conjugates, including HRP and poly-HRP antibody labeling kits, HRP conjugated to streptavidin, secondary antibodies, and small organic molecules as well as enzyme activity and quantitation assay kits. We also offer a wide selection of chromogenic, fluorogenic, and chemiluminescent HRP substrates to satisfy any experimental design or instrument setup.

 



HRP - A Robust Reporter Enzyme


The widespread adoption of HRP as an enzyme reporter system is primarily due to three factors. First, HRP has the ability to amplify weak signals and enhance the detectability of low abundance target molecules. Second, the relatively small size of HRP (~44 kDa) compared to other reporter enzymes such as alkaline phosphatase (~140 kDa) further improves intracellular penetration into samples, reduces steric hindrance, and minimizes immunoreactivity loss. Third, the high turnover rate and good stability of HRP enable rapid and strong signal generation when incubated with a proper substrate.

 

Poly-HRP Conjugates


MegaWox™ polyHRP-Streptavidin Conjugate is an enzymatic label consisting of an HRP heteropolymer core and a streptavidin shell. Similarly, MegaWox™ polyHRP-Protein A Conjugate, polyHRP-Goat Anti-Rabbit IgG Conjugate, and polyHRP-Goat Anti-Mouse IgG Conjugate are enzymatic labels consisting of an HRP heteropolymer core and either protein A, GAR, or GAM as target binding molecules. Additionally, Buccutite™ Poly-HRP Antibody Labeling kits enable the direct labeling of antibodies to polymers of HRP, resulting in conjugates that deliver the highest sensitivity and signal-to-noise ratios. Poly-HRP conjugates are ideal for applications where the target of interest is in low-abundance or when sample volume is limited.

Each molecule of a Poly-HRP conjugate contains a multitude of HRP (polymerized horseradish peroxidase), supplying more enzymatic activity per binding event and consequently a dramatic signal amplification in conventional assay applications. The amplification is achieved without major procedural modification, and the conjugate may be employed in a variety of assays, including ELISA, IHC, ICC, Western blotting, or DNA/RNA in situ hybridization.

Common Poly-HRP Conjugates Applications


The Poly-HRP-streptavidin conjugate can be used with virtually any biotin-labeled product, including biotinylated probes and primary and secondary antibodies. The conjugate is especially applicable to ultra-sensitive ELISA applications, wherein picogram- to- sub picogram per mL sensitivity is required. Alternatively, signal enhancement by the conjugate can be exploited to reduce the number and duration of processing steps required when performing conventional multistep assays, such as ELISA, without sacrificing overall assay sensitivity. Similarly, incorporation of a one-step poly-HRP conjugate method can shorten the IHC procedure by avoiding the two-step biotinylated secondary antibody and avidin-biotin complex (ABC) reagent step that is required using standard avidin-biotin systems.

 

Table 1. Product ordering information for MegaWox™ polyHRP conjugates

Product
Size
Cat No.
MegaWox™ polyHRP-Goat Anti-Mouse IgG Conjugate1 mg11035
MegaWox™ polyHRP-Goat Anti-Rabbit IgG Conjugate1 mg11037
MegaWox™ polyHRP-Protein A Conjugate1 mg11041
MegaWox™ polyHRP-Streptavidin Conjugate1 mg11039

 

Visualizing HRP and Poly-HRP Conjugates


HRP and poly-HRP conjugates are visualized using a substrate that, when oxidized by HRP using hydrogen peroxide, produces a quantifiable signal. Various HRP substrates have been developed and commercialized to exploit the desirable features of this enzyme detection system. These substrates fall into three general categories: chromogenic, fluorogenic or chemiluminescent substrates.
  1. Colorimetric substrates produce a visible color change that can be measured using an absorbance microplate reader. While they are the easiest to use and most affordable for HRP detection, chromogenic substrates are less-sensitive than fluorimetric or chemiluminescent substrates. They are ideal for applications where target abundance is high.
  2. Fluorimetric substrates produce highly fluorescent byproducts that can be measured using any fluorescence instrument. Offers greater sensitivity than colorimetric substrates.
  3. Chemiluminescent substrates generate luminescent byproducts that can be measured using a luminometer. Since the emission of a photon results from a chemical reaction and not light excitation, there is no background interference.

 

Table 2. Colorimetric, fluorimetric and chemiluminescent HRP Substrates.

Substrate
Detection
Absorbance (nm)
Ex (nm)
Em (nm)
Unit size
Cat No.
ABTSColorimetric420 nm--100 mL
1 L
11013
11001
TMBColorimetric450 nm / Yellow
650 nm / Blue
--100 mL
1 L
11012
11003
Amplite® BlueFluorimetric-324 nm409 nm25 mg11005
Amplite® ADHPFluorimetric-570 nm583 nm25 mg11000
Amplite® RedFluorimetric-570 nm583 nm1000 Assays11011
Amplite® IRFluorimetric-646 nm667 nm1 mg11009
LuminolChemiluminescent--410 nm1 mg11050

 

Buccutite™ HRP and Poly-HRP Antibody Labeling Kits


Buccutite™ Peroxidase (HRP) Antibody Labeling Kits enable researchers to efficiently label antibodies with HRP in less than 2 hours. The entire labeling process is completed in two simple mixing steps without subsequent column purification. Buccutite™ HRP conjugates are ideal for various immunoassay applications, including ELISA, IHC, and western blotting, and are highly stable for long-term storage. Buccutite™ Peroxidase Antibody Labeling Kits are available in three sizes optimized for labeling 25 µg, 100 µg, and 1 mg of antibody per labeling reaction.

For difficult applications that require an additional level of sensitivity, we offer Buccutite™ Poly-HRP Antibody Labeling kits. These kits enable the direct labeling of antibodies to polymers of HRP, resulting in conjugates that deliver the highest sensitivity and signal-to-noise ratios. Poly-HRP conjugates are ideal for applications where the target of interest is in low-abundance or when sample volume is limited.


Buccutite™ Peroxidase (HRP) Antibody Labeling Kit workflow (figure drawn in BioRender).

Key features of the Buccutite™ HRP and Poly-HRP Antibody Labeling Kits

  • Can effectively and efficiently label antibody concentrations as low as 100 µg/mL (SMCC requires at least 1 mg/mL)
  • Labeled antibodies are ready in under 2 hours, with as little as ~15 minutes of hands on-time
  • A simple two-step mixing protocol reduces batch-to-batch variation
  • No column purification step required
  • 100% conjugation yield
  • Conjugates stable for long-term storage (4 °C, 12 months)
  • Conjugates can be used for ELISA, IHC, blotting, Power Styramide Signal Amplification, and TSA Imaging
  • Offers flexibility in experimental design, HRP conjugates are compatible with colorimetric, fluorimetric, and chemiluminescent substrates

 

Table 3. Buccutite™ Peroxidase Antibody Labeling kits designed for labeling antibodies with HRP and poly-HRP.

Name
Labeling Size / Reaction
Size
Cat No.
Buccutite™ Peroxidase (HRP) Antibody Conjugation KitOptimized for Labeling 25 µg Protein2 Labelings5505
Buccutite™ Peroxidase (HRP) Antibody Conjugation KitOptimized for Labeling 100 µg Protein2 Labelings5503
Buccutite™ Peroxidase (HRP) Antibody Conjugation KitOptimized for Labeling 1 mg Protein1 Labeling5504
Buccutite™ Peroxidase (HRP) Antibody Conjugation KitOptimized for Labeling 1 mg Protein5 Labeling5504
Buccutite™ Poly-HRP Antibody Conjugation KitOptimized for Labeling 50 µg Protein1 Labeling5518
Buccutite™ Poly-HRP Antibody Conjugation KitOptimized for Labeling 50 µg Protein2 Labelings5519

 

Power Styramide™ Signal Amplification (PSA™) Technology



Sequential immunostaining of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma using iFluor® PSA™ Imaging kits. EpCam were labeled with rabbit anti-EpCam antibodies and iFluor® 488 PSA™ Imaging Kit with goat anti-rabbit IgG (Cat No. 45205), followed by washing. Pan-Keratin was labeled with mouse anti-pan Keratin antibodies and iFluor® 555 PSA™ Imaging Kit with goat anti-mouse IgG (Cat No. 45270). Nuclei were labeled with DAPI (Cat No. 17507). Images were acquired on a confocal microscope.
Power Styramide™ signal amplification (PSA™) is a highly sensitive catalyzed reporter deposition (CARD)-based technology commonly employed in the detection of low-abundance targets encountered in IHC, ICC, and ISH applications. The technology is based upon in situ HRP-catalyzed deposition of styramide onto and directly adjacent to a target molecule of interest (protein, carbohydrate, or nucleic acid). In the presence of low concentrations of hydrogen peroxide, HRP converts a labeled styramide substrate into a highly reactive species that covalently binds to accessible tyrosine residues on proteins in the vicinity of the HRP. This produces high concentration styramide labeling, resulting in exceptionally high sensitivity of this system for low abundance targets.

Power Styramide probes are available that have been labeled with iFluor® Styramide dyes, superior alternatives to Alexa Fluor® tyramide dyes. They are also available conjugated to a variety of haptens, including biotin, DNP, chloramphenicol, sulfathiazol, cinoxacin, fentanyl, danofloxacin, AOZ, and DIG. For multicolor detection applications, multiple rounds of styramide signal amplification can be performed using different Styramide Amplification Kits. By following the PSA™ deposition step with antibody removal in multiplex fluorescent IHC, PSA™ facilitates detection of the low-abundance targets and additionally simplifies antibody panel selection since primary antibodies may be used, regardless of host species or isotype.

 

Table 4. Available iFluor® PSA™ Imaging Kits

iFluor® PSA Imaging Kit
Secondary Antibody-HRP
Ex (nm)
Ex (nm)
Filter Set
Unit Size
Cat No.
iFluor® 350 PSA™ Imaging KitGoat Anti-Mouse IgG-HRP344448DAPI100 tests45250
iFluor® 350 PSA™ Imaging KitGoat Anti-Rabbit IgG-HRP344448DAPI100 tests45200
iFluor® 488 PSA™ Imaging KitGoat Anti-Mouse IgG-HRP491514FITC100 tests45260
iFluor® 488 PSA™ Imaging KitGoat Anti-Rabbit IgG-HRP491514FITC100 tests45205
iFluor® 555 PSA™ Imaging KitGoat Anti-Mouse IgG-HRP552567Cy3/TRITC100 tests45270
iFluor® 555 PSA™ Imaging KitGoat Anti-Rabbit IgG-HRP552567Cy3/TRITC100 tests45220
iFluor® 594 PSA™ Imaging KitGoat Anti-Mouse IgG-HRP592619Cy3/TRITC100 tests45280
iFluor® 594 PSA™ Imaging KitGoat Anti-Rabbit IgG-HRP592619Cy3/TRITC100 tests45230
iFluor® 647 PSA™ Imaging KitGoat Anti-Mouse IgG-HRP649665Cy5100 tests45240
iFluor® 647 PSA™ Imaging KitGoat Anti-Rabbit IgG-HRP649665Cy5100 tests45290