Signal Guard™ HRP reaction stopping reagent

HRP coupling reactions provide sensitive biomolecular assays based on hydrogen peroxide- generating enzyme systems linked to peroxidase- mediated oxidation. Fluorogenic HRP substrates are preferred to use for enhancing assay sensitivities. Among them, the most commonly used HRP substrates include ADHP (also called Amplex® Red, #11000), Amplex® UltraRed and Amplite® Red. Typically, detection reactions are performed in microplate wells and are initiated by adding a fluorogenic HRP substrate, resulting in a continuous fluorescence increase. It is critical to ensure that the timing of the standard and unknown sample measurements is the same. Our Signal Guard™ HRP reaction stopping reagent provides convenience and control by allowing the fluorescence signal-generating reaction to be terminated at a user-determined time point. After addition of the stop reagent, the fluorescence signal remains stable. The Signal Guard™ HRP reaction stopping reagent is designed for use in conjunction with ADHP (Amplex® Red ), Amplite® and Amplex®UltraRed fluorogenic substrates. Under the same conditions, Signal Guard™ HRP reaction stopping reagent significantly outperforms the Amplex® Red/UltraRed Stop Reagent (#A33855) from ThermoFisher. Our Signal Guard™ HRP reaction stopping reagent can also be used in other HRP reaction systems.

Example protocol

AT A GLANCE

Important Note

Thaw each kit component at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Signal Guard™ HRP Reaction Stopping Reagent (20X)

Add 500 µL of ddH2O to Signal Guard™ HRP reaction stopping reagent vial and mix well.

Note Aliquot in small quantites and store at -20 °C in dark plac and avoid light.

PREPARATION OF WORKING SOLUTION

Signal Guard™ HRP Reaction Stopping Reagent (1X)

Add 50 µL of Signal Guard™ HRP reaction stopping reagent into 950 µL ddH₂O and mix well.
Note Prepare the working solutions freshly as needed, and avoid light.

SAMPLE EXPERIMENTAL PROTOCOL

At the desired stopping time point, add 20 µL of Stop Reagent solution (1X) per 100 µL volume in each microplate well.

Note: For other reaction volumes, adjust the addition of 1X Stop Reagent proportionally (e.g., add 5 µL to a 25 µL reaction volume). The 1X stop reagent should be added to all wells, including any reagent controls without HRP.

Note: The time-dependent fluorescence signal increase will terminate immediately, and the fluorescence signal level should remain stable for at least 5 hours.

Citations

View all 9 citations: Citation Explorer

Assessment of Tofacitinib and Ruxolitinib and their Anti Inflammatory Effects on Myeloperoxidase
Authors: Milton, Amber
Journal: (2017)

Patterned Photonic Nitrocellulose for Pseudo-Paper ELISA
Authors: Chi, Junjie and Gao, Bingbing and Sun, Mi and Zhang, Fengling and Su, Enben and Liu, Hong and Gu, Zhongze
Journal: Analytical Chemistry (2017)

Myeloperoxidase--Hepatocyte--Stellate Cell Cross Talk Promotes Hepatocyte Injury and Fibrosis in Experimental Nonalcoholic Steatohepatitis
Authors: Pulli, Benjamin and Ali, Muhammad and Iwamoto, Yoshiko and Zeller, Matthias WG and Schob, Stefan and Linnoila, Jenny J and Chen, John W
Journal: Antioxidants & redox signaling (2015): 1255--1269

Myeloperoxidase Nuclear Imaging for Epileptogenesis
Authors: Zhang, Yinian and Seeburg, Daniel P and Pulli, Benjamin and Wojtkiewicz, Gregory R and Bure, Lionel and Atkinson, Wendy and Schob, Stefan and Iwamoto, Yoshiko and Ali, Muhammad and Zhang, Wei and others, undefined
Journal: Radiology (2015): 822--830

Ordered cleavage of myeloperoxidase ester bonds releases active site heme leading to inactivation of myeloperoxidase by benzoic acid hydrazide analogs
Authors: Huang, Jiansheng and Smith, Forrest and Panizzi, Peter
Journal: Archives of biochemistry and biophysics (2014): 74--85

References

View all 61 references: Citation Explorer

K+-independent actions of diazoxide question the role of inner membrane KATP channels in mitochondrial cytoprotective signaling
Authors: Drose S, Br and t U, Hanley PJ.
Journal: J Biol Chem (2006): 23733

Real-time assessment of spatial and temporal coupled catalysis within polyelectrolyte microcapsules containing coimmobilized glucose oxidase and peroxidase
Authors: Stein EW, Volodkin DV, McShane MJ, Sukhorukov GB.
Journal: Biomacromolecules (2006): 710

O2 Delivery and Redox State are Determinants of Compartment-specific Reactive Oxygen Species in Myocardial Reperfusion
Authors: Stoner JD, Clanton TL, Aune SE, Angelos MG.
Journal: Am J Physiol Heart Circ Physiol. (2006)

Fibrillar beta-amyloid peptide Abeta1-40 activates microglial proliferation via stimulating TNF-alpha release and H2O2 derived from NADPH oxidase: a cell culture study
Authors: Jekabsone A, M and er PK, Tickler A, Sharpe M, Brown GC.
Journal: J Neuroinflammation (2006): 24

Biochemical activity of reactive oxygen species scavengers do not predict retinal ganglion cell survival
Authors: Schlieve CR, Lieven CJ, Levin LA.
Journal: Invest Ophthalmol Vis Sci (2006): 3878

Page updated on November 21, 2024

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Catalog Number	11020
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Physical properties

Solvent

Water

Storage, safety and handling

Certificate of Origin	Download PDF
H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501