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
Biochem/physiol Actions
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.
Preparation Note
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.
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
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:
Refrigerate (4°C):
Sample Buffer
Assay Buffer
Wash Buffer
Substrate
Anti-Human TACE-coated 96-well plate
Deep Freeze (-70°C):
Control*
*Note: Following initial use the Control should be dispensed into aliquots and stored at -70°C. Avoid freeze/thaw cycles.
Analysis Note
Positive Control
Human recombinant TACE
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.
Other Notes
Anti-Human TACE-Coated 96-Well Plate, Control, Substrate, Sample Buffer, Assay Buffer, Wash Buffer, Plate Sealer, and a user protocol.
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 Citations
DasGupta S., et al. 2009. Bioorg. Med. Chem.17, 3604.
anti-human TACE-coated 96-well plate, TACE control, substrate, sample buffer, assay buffer, wash buffer, plate sealer, user protocol
Legal Information
CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
Toxicity: Multiple Toxicity Values, refer to MSDS (O)
