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Myeloperoxidase (MPO) Fluorometric Activity Assay Kit

based on 3 citations in multiple journalsMyeloperoxidase (MPO) Fluorometric Activity Assay Kit34.1 4
Highly Sensitive Assay, HTS
Catalog #: K745
$460.00

Product Details

Cat # +Size K745-100
Size 100 assays
Detection Method Fluorescence (Ex/Em = 485/525 nm)
Species Reactivity Mammalian
Applications N/A
Features & Benefits • Simple procedure
• Fast and convenient
• Sensitive assays for measuring MPO as low as 0.5 μU per well
Kit Components • Assay Buffer
• MPO Substrate Stock
• MPO Probe
• Fluorescein Standard (1 mM)
• MPO Positive Control
Storage Conditions -20°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.

Details

Myeloperoxidase (MPO) is a peroxidase enzyme (EC 1.11.1.7) most abundantly present in neutrophil granulocytes. It is a green hemoprotein found in neutrophils and monocytes that catalyzes the reaction of hydrogen peroxide and halide ions to form cytotoxic acids and other intermediates that play a role in the oxygen-dependent killing of tumor cells and microorganisms. Its heme pigment causes the green color in secretions rich in neutrophils, such as pus and some forms of mucus. Furthermore, it can oxidize tyrosine to a tyrosyl radical using hydrogen peroxide as an oxidizing agent. In BioVision’s MPO Assay Kit, MPO catalyzes the production of NaClO from H₂O₂ and NaCl. Subsequently, NaClO will react stoichiometrically with Aminophenyl fluorescein (APF) to generate fluorescein, which has a strong fluorescence and can be detected at Ex/Em = 485/525 nm. The kit provides a rapid, simple, sensitive, and reliable test suitable as a high throughput assay of MPO activity. This kit can be used to detect MPO activity as low as 0.5 μU per well.


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Can this kit be used with frozen cells/tissue?
We always prefer fresh samples for the most accurate results. It is possible to use frozen cells/tissue samples as long as they have been stored with minimum delay after collection and have been stored at -80C without freeze-thaw. However, the MPO enzyme is quite vulnerable to losing activity during storage. The functional enzyme contains disulphide bonds and if reduced, the enzyme can lose activity. It also contains heme which can get oxidized by the peroxide generated by MPO activity and the result is that the enzyme loses activity by auto-oxidation.
The same customer used K744 and K745 but got very different raw data for the increasing dilutions of the samples. Why?
It is very important to be able to distinguish the two kits by principle. For K744, the lower the OD, the higher the MPO activity. If you add too much sample, the OD will be so low; it could be below the detection limit of absorbance instruments. For K745, the higher the RFU, the higher the MPO activity. So, for this kit, adding too much sample can saturate the detector and the substrate can be limiting. This will result in discrepant differences between dilutions.
How much cells/tissue is needed for this assay?
There is no set amount of cells/tissue needed for this assay. The amount depends on how much active MPO is present in the samples. Typically 1-2 million cells and 10-100 mg tissue can be used per assay.
What is the activity of the positive control? How can the value be higher to compare with samples?
The positive control is only a benchmark sample. As long as the values are within the range of the std curve this is fine. The positive control is not be used to compare values with the samples. The positive control is provided to validate that the assay components are all working. If the values are low, the customer can add more volume to get higher values but this is not necessary as long as the values are within the std. curve range. MPO is a very vulnerable enzyme to freeze-thaw and can lose activity with storage over time.
What is the dilution factor used for?
If a certain volume of neat sample is added to the well and volume is made up with the assay buffer up to 50 ul, then dilution factor does not apply. If the sample is prediluted before adding to the well, then the dilution factor is used. For example, if 10 ul of a 5x diluted sample is used, then V=0.01 ml and Dilution factor =5.
What is the shelf-life of the kit? What about the positive control is stored unthawed at -20C or -80C for 2-3 years?
The shelf-life of the unopened kit is 1 year from the date of shipping from us when stored at -20C. Trypsin is notoriously well known for its self-destructive protease activity when stored for long periods of time. Once reconstituted, the positive control is to be used within 2months for best results. Hence we do not expect the positive control enzyme to retain normal activity over 2-3 years even at -20C or -80C.
How does the sensitivity compare between K744 and K745?
K745 is fluorometric and is at least 10 times more sentivie than K744. K744 standard curve range is 1-50 nmoles of TNB formed. K745 Standards range between 1-50 pmoles of Fluorescein.
Can individual components of this kit be purchased separately?
Yes, any of the kits components can be purchased separately without having to buy the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Are trial sizes of this kit available?
Unfortunately, we do not have trial sizes of this kit available. However,for customers based in the US or Canada, a 10% off list price introductory discount can be offered on the list price. For all other customers based out of this area our regional BioVision distributor should be contacted.
Can RIPA buffer be used to prepare samples for this kit?
For any enzyme assay, we do not recommend RIPA buffer since it contains SDS and this can denature proteins and affect enzyme activity. We have tested and recommend using our Assay bufefr provided in the kit for best results.
Ranganathan et al., CXCR2 knockout mice are protected against DSS-colitis-induced acute kidney injury and inflammation. Am J Physiol Renal Physiol, Nov 2013; 305: F1422 - F1427.
Punithavathi Ranganathan et al., Netrin-1 regulates colon-kidney cross talk through suppression of IL-6 function in a mouse model of DSS-colitis. Am J Physiol Renal Physiol, May 2013; 304: F1187 - F1197.
Churg, A. et. al. Late Intervention with a Myeloperoxidase Inhibitor Stops Progression of Experimental COPD. Am. J. Respir. Crit. Care Med., Oct 2011; 10.1164/rccm.201103-0468OC.
For more citations of this product click here