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48-602MAG
Buffer Detection Kit for Magnetic Beads
1 Kit
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Description
Overview
The InnoZyme™ Myeloperoxidase Activity Kit is a specific and sensitive assay designed to measure myeloperoxidase activity and to screen enzyme inhibitors. The assay uses a polyclonal antibody specific for human MPO immobilized on a 96-well plate to specifically capture the enzyme. Activity of captured myeloperoxidase is measured using a detection reagent that includes TMB and hydrogen peroxide. Following color development, the reaction is stopped with sulfuric acid and the absorbance of the oxidized TMB is detected at 450 nm.
Catalogue Number
CBA024
Brand Family
Calbiochem®
Synonyms
InnoZyme™ MPO Activity Kit
Application Data
Materials Required but Not Delivered
• Distilled H2O • Pipettors or multi-channel pipettor carefully calibrated to the target volume • 37°C incubator • Microplate reader capable of measuring absorbance at 450 nm
References
References
Weil, S.C., et al. 1988. Science 240, 790. Winterbourn, C.C., et al. 2000. Curr. Opin. Hematol. 7, 53. Brennan, M.L., et al. 2003. New Engl. J Med. 349, 1595. Zheng, L., et al. 2004. J. Biol. Chem. in press Vita, J.A., et al. 2004. Circulation, 110, 1134.
Product Information
Detection method
Colorimetric
Form
96 Tests
Format
96-well plate
Kit contains
Anti-MPO Coated 96-Well Plate, MPO Standard, TMB, Hydrogen Peroxide, Assay Buffer, Sample Buffer, Stop Solution, Plate Sealers, and a user protocol.
Toxic if swallowed. Causes severe burns. Irritating to eyes and skin. May cause sensitization by skin contact. Possible risk of impaired fertility.
S Phrase
S: 24-26-28.1-3-30-36/37/39-45
Avoid contact with skin. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. After contact with skin, wash immediately with plenty of water. Keep in a cool place. Never add water to this product. Wear suitable protective clothing, gloves and eye/face protection. In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
Product Usage Statements
Intended use
The Calbiochem® InnoZyme™ Myeloperoxidase Activity Kit is designed to measure human myeloperoxidase activity in cell lysates and biological samples and to screen enzyme inhibitors.
Storage and Shipping Information
Ship Code
Dry Ice Only
Toxicity
Multiple Toxicity Values, refer to MSDS
Hazardous Materials Attention:
Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
Storage
-20°C
Storage Conditions
Upon arrival, store the unopened kit at -20°C. All kit components, once opened, can be stored for up to 3 months under the following conditions:
Store at 4°C: TMB, Anti-MPO Coated 96-Well Plate, Assay Buffer, Stop Solution, and Sample Buffer Store at -20°C: Myeloperoxidase Standard, and Hydrogen Peroxide
Note: Following initial use the Myeloperoxidase Standard and the Hydrogen Peroxide should be dispensed into aliquots and stored at -20°C. Avoid freeze/thaw cycles.
Protect from Light
Protect from light
Avoid freeze/thaw
Avoid freeze/thaw
Do not freeze
Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit contains
Anti-MPO Coated 96-Well Plate, MPO Standard, TMB, Hydrogen Peroxide, Assay Buffer, Sample Buffer, Stop Solution, Plate Sealers, and a user protocol.
Upon arrival, store the unopened kit at -20°C. All kit components, once opened, can be stored for up to 3 months under the following conditions:
Store at 4°C: TMB, Anti-MPO Coated 96-Well Plate, Assay Buffer, Stop Solution, and Sample Buffer Store at -20°C: Myeloperoxidase Standard, and Hydrogen Peroxide
Note: Following initial use the Myeloperoxidase Standard and the Hydrogen Peroxide should be dispensed into aliquots and stored at -20°C. Avoid freeze/thaw cycles.
Intended use
The Calbiochem® InnoZyme™ Myeloperoxidase Activity Kit is designed to measure human myeloperoxidase activity in cell lysates and biological samples and to screen enzyme inhibitors.
Background
Myeloperoxidase (MPO) (EC 1.11.1.7) catalyzes the formation of hypochlorous acid (HOCl), a powerful oxidant formed from chloride ions and hydrogen peroxide. It is stored in the azurophilic granules of neutrophils and is released as a result of inflammation and phagocytosis, making it an indicator of neutrophil activation. The products resulting from MPO activity exhibit strong microbiocidal effects. It is also a major component of myelocytic precursor cells and is also used as a marker for acute leukemia. MPO is a hemoprotein that consists of two 53-kDa subunits, two 15-kDa subunits, and two prosthetic porphyrins that are an essential part of the catalytic cycle.
Historically the enzyme is considered the most important component of the anti-bacterial activity of phagocytes, but recent reports implicate MPO in roles outside the host immune system. Brennen, et al. identified MPO as a new marker of inflammation that is superior to existing markers for assessing cardiac risk, as the level of enzyme was found to correlate with short-term risk of acute cardiac events among emergency care patients with chest pain. Recent findings indicate that MPO may play a role in the pathology of atherogenesis through the oxidation of apolipoprotein A1 and formation of defective HDL and altering its ability to bind and remove free cholesterol from peripheral tissues. Finally, Vita, et al. reports that serum myeloperoxidase level is an independent indicator of endothelial cell dysfunction in humans.
Principles of the assay
The Calbiochem® InnoZyme™ Myeloperoxidase Activity Kit is a specific and sensitive assay for measuring active human MPO. The assay uses a polyclonal antibody specific for human MPO immobilized on a 96-well plate to specifically capture the enzyme. Activity of captured myeloperoxidase is measured using a detection reagent that includes TMB and hydrogen peroxide. Following color development, the reaction is stopped with sulfuric acid and the absorbance of the oxidized TMB is detected at 450 nm.
Materials provided
• Anti-MPO Coated 96-well Plate (Kit Componet No. JA7970-1EA): 1 polystyrene plate, 96 wells, supplied as twelve 8-well strips, coated with an antibody against human myeloperoxidase • Myeloperoxidase Standard (Kit Componet No. JA7971-1EA): 1 vial, supplied at 100 µg/ml • Hydrogen Peroxide (Kit Componet No. JA7973-50UL): 1 vial, 50 µl, 30% hydrogen peroxide, supplied as 100X • TMB (Kit Componet No. JA7972-1ML): 1 vial, 1 ml • Assay Buffer (Kit Componet No. JA7974-5ML): 1 vial, 5 ml • Sample Buffer (Kit Componet No. JA7975-25ML): 1 bottle, 25 ml, supplied as 20X • ELISA Stop Solution (Kit Componet No. JA1616-12ML): 1 vial, 12 ml, 2.5N H₂SO₄ • Plate Sealer (Kit Componet No. JB155-EA): 2 each
Materials Required but not provided
• Distilled H2O • Pipettors or multi-channel pipettor carefully calibrated to the target volume • 37°C incubator • Microplate reader capable of measuring absorbance at 450 nm
Preparation
Dilute samples with Sample Buffer (1X) as needed. Note: If sample dilution is not required for sensitivity reasons, prepare the sample by adding 475 µl sample to 25 µl 20X Sample Buffer to ensure appropriate pH for the first incubation.
Reagent preparation
All reagents necessary to perform the assay are supplied with the kit. Equilibrate all reagents to room temperature (15-25°C) just prior to use.Sample Buffer (1X): Dilute Sample Buffer 1:20 by adding 25 ml Sample Buffer to 475 ml dH2O.
Myeloperoxidase Standard (1µg/ml): Dilute the Myeloperoxidase Standard 1:100 by adding 5 µl Myeloperoxidase Standard to 495 µl chilled Sample Buffer (1X) obtain a concentration of 1 µg/ml. Maintain the diluted Myeloperoxidase Standard
(1 µg/ml) on ice.
Note: Following initial thaw the Myeloperoxidase Standard should be dispensed into aliquots and stored at -20°C. Avoid freeze/thaw cycles.
Table 1: Myeloperoxidase Standard Dilutions
Prepare Myeloperoxidase Standard dilutions in the range of 5-100 ng/ml using the above guidelines.
Hydrogen Peroxide (0.3%): Dilute the Hydrogen Peroxide (30%) 1:100 by adding 5 µl Hydrogen Peroxide (30%) to 495 µl chilled dH2O.
Note: A total of 40 µl is required to cover the entire Coated 96-Well Plate. If the entire plate is not to be used at one time, prepare fresh Hydrogen Peroxide (0.3%) each time the assay is performed. Following initial thaw the Hydrogen Peroxide should be dispensed into aliquots and stored at -20°C. Avoid freeze/thaw cycles.
TMB Detection Reagent:Note: The desired amount of TMB Detection Reagent should be prepared fresh, just prior to use. To prepare TMB Detection Reagent, mix the following components:
• 7.16 ml dH2O
• 2.00 ml Assay Buffer
• 0.80 ml TMB
• 40 µl H2O2(0.3%)
Vortex gently 3 X 3 s
To prepare TMB Detection Reagent for a variable number of strips, use the following guidelines:
Table 2: Preparation of TMB Detection Reagent
To prepare TMB Detection Reagent for a variable number of strips, use the above guidelines.
Detailed protocol
It is recommended that all samples and standards be assayed in duplicate.
1. Remove the desired number of strips from the Anti-MPO Coated 96-Well Plate, return the remaining strips to foil pouch, and close the re-sealable edge. 2. Add 100 µl Myeloperoxidase Standard dilutions and samples to designated wells. The Blank should contain 100 µl Sample Buffer (1X). If inhibitor is to be included in the assay, add the desired amount of inhibitor to designated wells containing Myeloperoxidase Standard or sample; the total volume, including MPO and inhibitor should be 100 µl. 3. Cover the plate with the plate sealer and incubate 1 h at room temperature with gentle shaking. 4. Wash the plate four times with 400 µl Sample Buffer (1X). Following each wash discard the contents of the wells by inverting the plate over the sink; tap the plate on a paper towel to remove residual liquid. 5. Add 100 µl TMB Working Solution to each well and incubate 20-30 min at 37°C. 6. Add 100 µl ELISA Stop Solution to each well to stop the color development. 7. Read the absorbance at 450 nm.
Sensitivity
330 pg/ml
Sensitivity Notes
The minimum detectable activity, determined as the concentration of MPO at two standard deviations above the mean values of ten samples containing only diluent is 330 pg/ml.
Figure 1: MPO Standard Curve
Figure 2: Myeloperoxidase Activity in Biologicals Samples With and Without Inhibitor
MPO activity was measured in the presence of 20 µM ABAH (4-aminobenzoyl hydrazide), an irreversible inhibitor of myeloperoxidase. Cells were cultured in RPMI 1600 medium supplemented with 10% FCS and harvested at 80-90% confluency. Total cell lysates were prepared by treating cell pellets with CytoBuster™ Protein Extraction Reagent (see related products) according to the manufacturer's recommended protocol. Normal EDTA and heparin plasma and CSF were acquired from a commercial supplier. The myeloperoxidase activity assay was performed according to the Detailed Protocol.
Assay Range
5 -100 ng/ml
Plate configuration
Table 3: Sample Plate Layout
The above template may be used as a guide for setting up the Anti-MPO Coated 96-Well Plate.
Registered Trademarks
Calbiochem® is a registered trademark of EMD Chemicals, Inc. CytoBuster™, Interactive Pathways™, and InnoZyme™ are trademarks of EMD Chemicals, Inc.
