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Fluoro MPOHOCL

Fluoro MPOHOCL Cell Technology Fluorescent Enzymatic Assays
Fluoro MPOHOCL
Fluorescent Myeloperoxidase Chlorination and peroxidation Detection Kit

$575.00

in stock
Product code: MPOHOCL100
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Product Description

Key Benefits

  • Can monitor multiple time points to follow kinetics.
  • One-step, no wash assay.
  • Adaptable for High Throughput format.
  • Highly Sensitive.
  • Applications – Fluorescent Plate Reader.

Assay Principle

Myeloperoxidase (MPO) is a highly cationic glycosolated hemoprotein that has a molecular weight of 144kD. The hemoprotein consists of two dimers linked via a disulfide bridge. Each dimmer is composed of a heavy (53kD) and light (15kD) subunit. Each heavy chain contains an independently acting protoporphyrin group containing a central iron (1-5). MPO is present in the azurophilic granules of polymorphonuclear leukocytes (PMNs) and is unique to neutrophils and monocytes. However, monocytes contain only one third of the MPO found in PMN’s. MPO utilizes H202 produced by the neutrophils to oxidize a varity of aromatic compounds to give substrate radicals for bactericidal activity (4 review). This enzyme is unique however in that it can oxidize chloride ions to produce a strong nonradical oxidant,HOCl. HOCl is the most powerful bactericidal produced by neutrophils (4 review). Excessive production of these radicals can cause oxidative stress leading to oxidative tissue injury.

Chlorination Reaction:

H2O2 + MPO + Cl- —-> HOCl + APF (non fluorescent) —-> Fluorescent Dye

Excitation:488nm; Emission: 515-530nm

Peroxidation Reaction:

H2O2 + Detection reagent (non-fluorescent)+ MPO —–> fluorescent analog

Excitation 530-571nm Emission 590-600nm

Figure 1. In this figure, the MPO standard curve was serially diluted in 1X Reaction Buffer. Reaction cocktail (RC) was prepared as described (without EPO inhibitor). Next 50µL of MPO standard and 50µL of RC was added to individual well of 96- well black plates. The plate was incubated at room and temperature in the dark. Data collected Ex:530nm, Em:590nm

Figure 2. Red Blood Cell AChE (RBC-AChE) was purified and protein concentration determined using the BCA Protein Assay Kit (Pierce). The RBC-AChE was titrated in 1X reaction buffer and activity determined using the Fluoro: AChE kit. Acetylcholine concentration = 1mM final. In the graph the background value has been subtracted (0 RBC-AChE) to generate standard curve.

Applications

  • Detection of MPO chlorination activity in neutrophils and macrophages.
  • Detection of PMN infiltration in tissue samples (inflammation and innate host defense mechanisms).
  • Acute and chronic inflammatory disorders due to chlorination tissue damage.
  • MPO chlorination activity in acute and chronic manifestations of cardiovascular disease.

References

  • Olsen, R. L. & Little, c. (1983) Biochem. J. 209, 781-787.
  • Nauseef, W. M., and Malech, H. L. (1986) Blood, 67, 1504-1507.
  • Andrews, P. C., Parnes, C., and Krinsky, N. I. (1984) Arch. Biochem. Biophys., 228, 439-442.
  • Mark B. Hampton, Anthony J. Kettle, and Christine C. Winterbourn. Inside the Neutrophil Phagosome: Oxidants, Myeloperoxidase, and Bacterial Killing. Blood, Vol. 92 No. 9 (November 1), 1998: pp. 3007-3017
  • Mark B. Hampton, Anthony J. Kettle, and Christine C. Winterbourn. Inside the Neutrophil Phagosome: Oxidants, Myeloperoxidase, and Bacterial Killing. Blood, Vol. 92 No. 9 (November 1), 1998: pp. 3007-3017
  • Andrews, P.C., Parnes, C. & Krinsky, N.I. (1984) Comparison of myeloperoxidase and hemi-myeloperoxidase with respect to catalysis, regulation, and bacterial activity. Arch. Biochem. Biophys. 228, 439–442.
  • Bolognesi ML et al. Propidium-based polyamine ligands as potent inhibitors of acetylcholinesterase and acetylcholinesterase-induced amyloid-beta aggregation. J Med Chem. 2005 Jan 13;48(1):24-7.
  • Ken-ichi Setsukinai, Yasuteru Urano, Katsuko Kakinuma, Hideyuki J. Majima , and Tetsuo Nagano. Development of Novel Fluorescence Probes That Can Reliably Detect Reactive Oxygen Species and Distinguish Specific Species. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 5, Issue of January 31, pp. 3170–3175, 2003

Kit contents and Long Term storage

ReagentPart#Temperature
Detection Reagent, 1 VialPart# 4007-20C
10X Assay Buffer, 60mLPart# 30022-8C
Hydrogen Peroxide, 1000µL of a Stabilized 3% SolutionPart# 30122-8C
Myeloperoxidase, 1 Vial at 30Units/mLPart# 60152-8C
APF, 1 VialPart# 40112-8C
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