Analysis Note
The results are displayed in relative fluorescence units (RFU): 1. Correct the fluorescence from each sample and Control by subtracting the fluorescence of the Blank.2. Calculate the mean fluorescence for each sample and Control from duplicate readings to obtain the final RFU.
Positive ControlHuman recombinant TACE
Components
Anti-Human TACE-Coated 96-Well Plate, Control, Substrate, Sample Buffer, Assay Buffer, Wash Buffer, Plate Sealer, and a user protocol.
anti-human TACE-coated 96-well plate, TACE control, substrate, sample buffer, assay buffer, wash buffer, plate sealer, user protocol
General description
The InnoZyme™ TACE Activity Kit is a specific and sensitive assay designed to measure human TACE activity in cell lysates and biological samples and for screening enzyme inhibitors. An Anti-Human TACE-Coated 96-Well Plate is pre-coated with a monoclonal antibody specific for human TACE that captures the enzyme. Unbound material is discarded, the plate is washed, and the activity of captured TACE is measured using an internally quenched fluorescent substrate, MCA-KPLGL-Dpa-AR-NH2. Cleavage of the scissile amide bond, G-L, releases the fluorophore from the quenching molecule, Dpa, resulting in an increase in fluorescence. Fluorescence of the cleaved product, MCA-KPLG, is measured at an excitation wavelength of ~324 nm and emission wavelength of ~400 nm. The level of fluorescence is directly related to the enzyme activity. Highly specific: based on immunocapture of human TACE Highly sensitive and quantitative: assay range 5-100 ng/ml Convenient: 96-well format and non-radioactive detection Versatile: appropriate for measuring active TACE and high-throughput screening of TACE inhibitors
Legal Information
CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany
Other Notes
Kirkegaard, T., et al. 2004. Clin. Exp. Immunol.135, 146.Neumann, U., et al. 2004. Anal. Biochem.328, 166.Skovronsky, D.M., et al. 2001. J. Neurobiol.49, 40.Black, R.A., et al. 1997. Nature385, 729.Moss, M.L., et al. 1997. Nature385, 733.Selected CitationsDasGupta S., et al. 2009. ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum', window.location);" rel="nofollow" href="http://www.ncbi.nlm.nih.gov/pubmed/19394232→ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum">Bioorg. Med. Chem.17, 3604.
Packaging
1 kit in Glass bottle
Preparation Note
Dilute samples as necessary with Sample Buffer.Recommended guidelines for sample preparation:1. Total protein concentration in cell lysates should be >5 mg/ml2. Dilution factor for cell lysates at >5 mg/ml is approximately 1:20 or higher
All reagents necessary to perform the assay are supplied with the kit. Warm all reagents to room temperature (15-25°C) immediately prior to use.For analyzing biological samples the following reagent preparation is required: