Description
General description
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.
Biochem/physiol Actions
The Calbiochem InnoZyme Myeloperoxidase Activity Kit is designed to measure human myeloperoxidase activity in cell lysates and biological samples and to screen enzyme inhibitors.
Features and Benefits
Assay Time: 2 h
Preparation Note
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 (1g/ml): Dilute the Myeloperoxidase Standard 1:100 by adding 5 l Myeloperoxidase Standard to 495 l chilled Sample Buffer (1X) obtain a concentration of 1 g/ml. Maintain the diluted Myeloperoxidase Standard (1 g/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 l Hydrogen Peroxide (30%) to 495 l chilled dH2O. Note: A total of 40 l 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 l H2O2(0.3%)
Vortex gently 3 X 3 sTo 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.
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.
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.
Analysis Note
Positive Control
Human myeloperoxidase
Other Notes
Anti-MPO Coated 96-Well Plate, MPO Standard, TMB, Hydrogen Peroxide, Assay Buffer, Sample Buffer, Stop Solution, Plate Sealers, and a user protocol.
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.
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.
Legal Information
CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
Toxicity: Multiple Toxicity Values, refer to MSDS (O)
