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: Wash Buffer (1X): Dilute Wash Buffer 1:20 with dH₂O . To prepare Wash Buffer (1X) for a single wash step for the entire plate, add 2 ml Wash Buffer to 38 ml dH₂O. Control: Dilute the Control with chilled Sample Buffer. Refer to the vial label for lot-specific dilution information.Important Note: Following initial thaw the Control should be dispensed into aliquots and stored at -70°C. Avoid freeze/thaw cycles. Substrate (1X): Dilute Substrate 1:200 with Assay Buffer. To prepare Substrate (1X) for the entire plate, add 50 µl Substrate to 9.95 ml Assay Buffer.For inhibitor screening the following additional reagent preparation is required: Test inhibitor(s): Prepare dilutions of test inhibitors in Assay Buffer as necessary. Diluted Substrate: Dilute Substrate 1:50 with Assay Buffer. To prepare Substrate for the entire plate add 40 µl Substrate to 1.95 ml Assay Buffer.

Principle

The InnoZyme™ TACE Activity Kit is designed to measure human TACE activity in cell lysates and biological samples. The assay may also be used for screening enzyme inhibitors, but the end-user must supply purified enzyme for this application.

Storage and Stability

Upon receipt the unopened kit should be stored at -20°C or -70°C. All kit components, once opened, can be stored for up to 3 months under the following conditions:r>r>Refrigerate (4°C):r>Sample Bufferr>Assay Bufferr>Wash Bufferr>Substrater>Anti-Human TACE-coated 96-well plater>r>Deep Freeze (-70°C):r>Control*r>r>*Note: Following initial use the Control should be dispensed into aliquots and stored at -70°C. Avoid freeze/thaw cycles.

Warning

Toxicity: Multiple Toxicity Values, refer to MSDS (O)