Peroxidase from horseradish hrp

Manufactured by Merck Group

Sourced in United States

Peroxidase from horseradish (HRP) is a widely used enzyme in various laboratory applications. It catalyzes the oxidation of substrates in the presence of hydrogen peroxide, making it a valuable tool for a range of analytical and diagnostic procedures. The core function of HRP is to facilitate these oxidation reactions, which can be leveraged in various experimental and testing contexts.

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8 protocols using peroxidase from horseradish hrp

Synthesis and Characterization of Nanoparticles

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Chemicals that were used
in the study were purchased from different sources: CuCl₂·2H₂O, glutaraldehyde (GLA, 25 wt % in water), tri-sodium
citrate 5,5-hydrate, FeCl₃·6H₂O, FeSO₄·7H₂O, d-(+)-glucose (Glu), phenol,
Na₂HPO₄·2H₂O, and NaH₂PO₄·2H₂O were purchased from Merck; uric
acid (UA), (+)-catechin hydrate (CAT), bilirubin (Bil), caffeic acid
(CFA), fetal bovine serum (FBS), quercetin (QR), catalase from bovine
liver, uricase (UOx) from Candida sp., glucose oxidase
(GOx) from Aspergillus niger, choline
oxidase (ChOx) from Alcaligenes sp., peroxidase from
horseradish (HRP), choline chloride (ChCl), glycine, neocuproine (Nc)
hydrochloride hydrate, and trizma base were purchased from Sigma;
(3-aminopropyl)triethoxysilane (APTS), N-acetyl-l-cysteine (NAC), tetraethyl orthosilicate (TEOS), urea, 4-aminoantipyrine
(4-AAP), ascorbic acid (AA), gallic acid monohydrate (GA), l-glutathione reduced (GSH) were from Sigma-Aldrich; and trans-ferulic acid (FA), (R)-(+)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (trolox) (TR), and l-cysteine (CYS) were from Aldrich.

Avan A.N., Demirci-Çekiç S, & Apak R. (2022). Colorimetric Nanobiosensor Design for Determining Oxidase Enzyme Substrates in Food and Biological Samples. ACS Omega, 7(48), 44372-44382.

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Glyphosate HS ELISA Kit Protocol

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Anti-glyphosate-IgG modified magnetic beads (MBs) and HRP-conjugated-glyphosate (tracer) were obtained from ABRAXIS LLC (Warminster, PA, USA). The diluent solution (distilled water with preservative and stabilizers), washing solution (preserved deionized water), substrate solution (ready-to-use mixture of TMB and H₂O₂ in an organic medium, pH 6; concentrations not specified by the provider), and the Glyphosate HS Derivatization Kit were provided with the Glyphosate HS ELISA Kit by ABRAXIS LLC.
Glyphosate (certified reference material (CRM), EPA 547 glyphosate solution), 1 g/L in deionized water, chromatographic purity 99.9%, was obtained from Supelco (Milan, Italy). The CRM solution was stored at 4 °C until use. Solutions at different concentrations were freshly prepared by serial dilution from the CRM solution, stored at 4 °C, and used within the same day of preparation.
Peroxidase from horseradish (HRP) was purchased from Sigma-Aldrich-Fluka (Milan, Italy). Potassium chloride, potassium dihydrogenphosphate, and other chemicals were acquired from Merck (Milan, Italy).
Water from the Milli-Q Water Purification System (Milipore, Feltham, UK), was used for all solution buffers.

Bettazzi F., Romero Natale A., Torres E, & Palchetti I. (2018). Glyphosate Determination by Coupling an Immuno-Magnetic Assay with Electrochemical Sensors. Sensors (Basel, Switzerland), 18(9), 2965.

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Electrochemical Detection of Ochratoxin A

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Analytical standards of mycotoxins, bovine serum albumin (BSA), and peroxidase from horseradish (HRP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The OTA monoclonal antibody 2D8 (mAb, 2D8) was prepared in our laboratory [32 (link)]. The activated peroxidase protein labeling kit (EL0156) was purchased from Huzhou InnoReagents Co., Ltd. (Huzhou, China). Magnetic nanoparticles (M-270, 14305D) were purchased from Invitrogen (Carlsbad, CA, USA). Screen-printed electrodes based on carbon working and counter electrodes were provided by Prof. P. Wang, Biosensor National Special Laboratory, Zhejiang University. Other reagents of analytical grade, including hydrogen peroxide (30%) (H₂O₂), hydroquinone (HQ), and benzoquinone (BQ) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

Zhang X., Wang Z., Xie H., Sun R., Cao T., Paudyal N., Fang W, & Song H. (2018). Development of a Magnetic Nanoparticles-Based Screen-Printed Electrodes (MNPs-SPEs) Biosensor for the Quantification of Ochratoxin A in Cereal and Feed Samples. Toxins, 10(8), 317.

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Luciferase-Based Luminescence Assay

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A stock solution of luciferase (product number: L9420), 1, 4-dithiothreitol (DTT), adenosine 5’-triphosphate disodium salt (ATP), coenzyme A sodium salt (CoA), BSA, ovalbumin (OVA), luminol sodium salt, 4-(imidazol-1-yl)phenol (4-IMP), hydrogen peroxide solution (H₂O₂), peroxidase from horseradish (HRP) and thrombin were purchased from Sigma-Aldrich (St. Louis, MO, USA). D-luciferin sodium salt was purchased from J&K Scientific (Beijing, China). Collagen was purchased from Hyphen Biomed (Neuville-sur-Oise, France). Hank’s balanced salt solution (HBSS) was obtained from ThermoFisher Scientific (Waltham, MA, USA). Ly294002 (phosphoinositite 3 kinase (PI3K) inhibitor) and Staurosporine (protein kinase C (PKC) inhibitor) were purchased from Abmole Bioscience (Houston, TX, USA). Prostaglandin E1 (PGE1) and other commonly used chemical reagents were purchased from Aladdin Industrial (Shanghai, China).

Wang L., Li Y., Guo R., Li S., Chang A., Zhu Z, & Tu P. (2019). Optimized bioluminescence analysis of adenosine triphosphate (ATP) released by platelets and its application in the high throughput screening of platelet inhibitors. PLoS ONE, 14(10), e0223096.

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Synthesis and Characterization of NIPAm-Based Polymers

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N-Isopropylacrylamide (NIPAm) (Sigma-Aldrich, 99%) was recrystallized from a mixture of n-hexane/toluene (4 : 1 v/v). N-2-thiolactoneacrylamide (TlaAm) was synthesized according to a procedure described in a previous publication.^{8 (link)}N,N′-methylenebisacrylamide (Bis) (Sigma-Aldrich, 99%), N,N,N′,N′-tetramethylethylenediamine (TEMED) (Carl Roth, ≥99%), potassium persulfate (Sigma-Aldrich, ≥99.0%), nicotinamide adenine dinucleotide disodium salt (NADH) (Roche, ∼100%, grade I), 2-deoxy-d-ribose-5-phosphate sodium salt (DRP) (Sigma, ≥95%), glucose (Sigma-Aldrich, ≥99.5%), dimethyl sulfoxide (DMSO) (Carl Roth, ≥99.8%, <100 ppm H₂O), deuterated chloroform (Carl Roth, 99.8% D), Atto-665-maleimide (Atto-Tec), bovine serum albumin (BSA) (Sigma-Aldrich, >98%, lyophilized powder, heat shock fraction), peroxidase from horseradish (HRP) (Sigma-Aldrich), (lyophilized powder, 150 U mg⁻¹), glucose oxidase from Aspergillus niger (Carl Roth, lyophilized powder, 280 U mg⁻¹) and α-glycerophosphate dehydrogenase-triosephosphate isomerase from rabbit muscle (Sigma-Aldrich), Type III, ammonium sulfate suspension, TPI 750–2000 units per mg protein, GDH 75–200 units per mg protein (biuret) were used as received.

Reinicke S., Fischer T., Bramski J., Pietruszka J, & Böker A. (2019). Biocatalytically active microgels by precipitation polymerization of N-isopropyl acrylamide in the presence of an enzyme. RSC Advances, 9(49), 28377-28386.

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Horseradish Peroxidase ABTS Assay

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Peroxidase from horseradish HRP was purchased from Sigma (St. Louis, MO, USA). 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) was purchased from Sigma. Disodium hydrogen orthophosphate (Na₂HPO₄), citric acid and hydrogen peroxide (H₂O₂) were purchased from Reakhim (Moscow, Russia). All solutions were prepared using deionized ultrapure water (with 18.2 MΩ×cm resistivity) obtained with a Simplicity UV system (Millipore, Molsheim, France).

Ivanov Y.D., Pleshakova T.O., Shumov I.D., Kozlov A.F., Ivanova I.A., Valueva A.A., Tatur V.Y., Smelov M.V., Ivanova N.D, & Ziborov V.S. (2020). AFM Imaging of Protein Aggregation in Studying the Impact of Knotted Electromagnetic Field on A Peroxidase. Scientific Reports, 10, 9022.

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Grapevine Cell Elicitation with B. cinerea

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Azure A (AzA) (80%), chloroplatinic acid hexahydrate (≥99.9%), citric acid (trisodium salt), L-dehydroascorbic acid (DHA), ethanol (≥ 99.5% v/v), D(+)-glucose, H2O2 (35%), D-mannitol, L-methionine, trans-resveratrol, salicylic acid, sodium Lascorbate, sodium lauryl sulfate (SDS 95%), ascorbate oxidase (AO) from Cucurbita sp.
(156.60 units/mg solid), catalase (CAT) from bovine liver (2,200 units/mg protein) and peroxidase from horseradish (HRP, 313 units/mg solid) were obtained from Merck-Sigma-Aldrich (Spain). MeJa was purchased from Duchefa (Spain). KCl, KNO3, K2HPO4, KH2PO4 and K4Fe(CN)6 were acquired from Merck. H2SO4 and HCl were supplied by Panreac. Clopyralid 72% came from Dow AgroSciences (US).
The fungus B. cinerea was grown on potato dextrose agar plates for 20 days at 28 ºC until extensive sporulation. Spores were recovered by adding 10 ml of distilled sterile water to each plate. The suspension was filtered through nylon mesh in order to remove contaminating hyphae. A haemocytometer was used to determine the spore concentration.
For the elicitation experiments, grapevine cell cultures were cultivated with B. cinerea spores at a final concentration of 6 × 10 4 spores/mL. All the measurements were taken with freshly prepared solutions. The concentration of the H2O2 stock solutions was spectrophotometrically checked at 240 nm (ε = 43.6 M -1 cm -1 ) [28] .

Jiménez-Pérez R., Almagro L., González-Sánchez M.I., Pedreño M.Á, & Valero E. (2020). Non-enzymatic screen-printed sensor based on PtNPs@polyazure A for the real-time tracking of the H₂O₂ secreted from living plant cells. Bioelectrochemistry (Amsterdam, Netherlands), 134.

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Glucose Oxidase-based Biosensing Protocol

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Phosphate buffered saline (PBS) (Sigma Aldrich, USA), glucose oxidase from Aspergillus niger (211 U/mg) (Sigma Aldrich, USA), peroxidase from horseradish (HRP, 325 U/mg) (Sigma Aldrich, USA), potassium chloride (KCl) (Sigma Aldrich, USA), potassium iodide (KI) (Sigma Aldrich, USA), chitosan-low molecular weight (Sigma Aldrich, USA), acetic acid-2% (v/v) in H2O (Sigma Aldrich, USA), D(+)-glucose (C6H12O6) -≥99.5% (GC) (Sigma Aldrich, USA), paper-towel (Eagle photocell towel, Eagle Professional, Turkey), Self-inking stamp (Kas ¸em, Turkey), methacrylic/acrylic resin (HTM140 V2, EnvisionTEC, USA), thermoplastic filament (Z-ABS, Zortrax, USA), Whatman qualitative filter paper-grade 1 (Sigma Aldrich, USA), artificial saliva (NeutraSal, Germany).

Gölcez T., Kiliç V, & Sen M. (2021). A Portable Smartphone-based Platform with an Offline Image-processing Tool for the Rapid Paper-based Colorimetric Detection of Glucose in Artificial Saliva. Analytical sciences : the international journal of the Japan Society for Analytical Chemistry, 37(4).

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