InnoZyme TACE Activity Kit Cat. No. CBA042

Transcription

1 User Protocol CBA042 Rev. 26 January 2006 JSW Page 1 of 5 InnoZyme TACE Activity Kit Cat. No. CBA042 Note that this user protocol is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions. Full details are available at. Table of Contents Page Storage 1 Intended Use 1 Background 1 Principle of the Assay 2 Materials Provided 2 Materials Required but not Provided 2 Reagent Preparation 2 Detailed Protocol 3 Calculation of Results 4 Assay Characteristics and Examples 4 References 5 Related Products 5 Storage 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. Intended Use The InnoZyme TM TACE Activity Kit is designed to measure human TACE activity in cell lysates and biological samples and for screening enzyme inhibitors. Background TACE (TNF-α Converting Enzyme), also known as ADAM17 or α-secretase, is a membrane-anchored zinc protease expressed in its active form on the surface of several cell types. The enzyme belongs to the ADAM family of proteins, which are multi-domain proteins consisting of a signal sequence, a prodomain, metalloprotease-like, disintegrin-like, cysteine-rich, and EGF-like repeat domains, followed by a transmembrane region and

2 User Protocol CBA042 Rev. 26 January 2006 JSW Page 2 of 5 cytoplasmic tail. The most well known physiological substrate for TACE is pro-tnf-α, the membrane-bound precursor of TNF-α, which is a potent pro-inflammatory cytokine. Active TNF-α is released from the membrane and mediates the recruitment and activation of inflammatory cells to injured or infected tissues. Elevated levels of circulating TNF-α have been demonstrated in several acute and chronic pathological conditions, such as LPSinduced septic shock, arthritis, pleurisy, Crohn s disease, and inflammatory bowel disease. TACE is also responsible for the proteolytic cleavage of amyloid precursor protein (APP), the protein from which amyloid β- peptides are derived. β-peptides are among the major components of senile plaques, the aberrant structures that are present in the brain of Alzheimer s disease (AD) patients. TACE was found to localize with senile plaques and neurofibrillary tangles in the hippocampus and cortex of brains from AD patients. In addition to pro-tnf-α and APP, TACE is also known to cleave p55, p75 TGF-α, interleukin-1r-ii, and L-selectin. Principle of the Assay The InnoZyme TM TACE Activity Kit is a specific and sensitive assay for measuring active human TACE. The 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-NH 2. 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. Materials Provided Anti-Human TACE-Coated 96-Well Plate (Kit Component No. JA9118): 96-well polystyrene plate supplied as 6 strips, 2 X 8 wells each, coated with an antibody specific for human TACE, 1 each Control (Kit Component No. JA9119): human recombinant TACE, 1 vial Substrate (Kit Component No. JA9120): MCA-KPLGL-Dpa-AR-NH 2, 60 µl, 2 mm in DMSO, 1 vial Sample Buffer (Kit Component JA9122): 20 ml, 2 bottles Assay Buffer (Kit Component JA9121): 20 ml, 1 bottle Wash Buffer (Kit Component No. JA9123): 20 ml, supplied as 20X, 1 bottle Plate Sealer (Kit Component No. JA9124): 1 each Materials Required but not Provided Distilled H 2 O Pipettors or multi-channel pipettor precisely calibrated to the target volume 37 C incubator Fluorescent plate reader capable of measuring fluorescence at excitation nm and emission nm Reagent Preparation All reagents necessary to perform the assay are supplied with the kit. Warm all reagents to room temperature ( C) immediately prior to use. For analyzing biological samples the following reagent preparation is required: Wash Buffer (1X): Dilute Wash Buffer 1:20 with dh 2 O Example: To prepare Wash Buffer (1X) for a single wash step for the entire plate, add 2 ml Wash Buffer to 38 ml dh 2 O.

3 User Protocol CBA042 Rev. 26 January 2006 JSW Page 3 of 5 Control Dilute the Control with chilled Sample Buffer. Refer to the vial label for lot-specific dilution information. Samples Important Note: Following initial thaw the Control should be dispensed into aliquots and stored at 70 C. Avoid freeze/thaw cycles. Dilute samples as necessary with Sample Buffer. Recommended guidelines for sample preparation: Total protein concentration in cell lysates should be > 5 mg/ml Dilution factor for cell lysates at > 5 mg/ml is approximately 1:20 or higher Substrate (1X) Dilute Substrate 1:200 with Assay Buffer Example: 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 Example: To prepare Substrate for the entire plate add 40 µl Substrate to 1.95 ml Assay Buffer. Detailed Protocol Note: It is recommended that all samples and controls be assayed in duplicate. Recommended Protocol for Assessing Activity in Biological Samples 1. Remove the desired number of strips from the Anti-Human TACE-Coated 96-Well Plate. Return the remaining strips to the re-sealable foil pouch, seal the edge, and store at +4 C. 2. Wash the plate by adding µl Wash Buffer (1X) to each well. Following the wash discard the contents of the wells by shaking the contents of the wells into the sink; invert the plate and tap on a paper towel to remove residual liquid. Repeat for a total of 2 washes. 3. Add 100 µl Control and samples to designated wells; prepare a Blank by adding 100 µl Sample Buffer to designated wells. 4. Cover the plate with the Plate Sealer and incubate 1 h at room temperature with gentle shaking. 5. Wash the plate by adding µl Wash Buffer (1X) to each well. Following the wash discard the contents of the wells by shaking the contents of the wells into the sink; invert the plate and tap on a paper towel to remove residual liquid. Repeat for a total of 5 washes. 6. Add 100 µl Substrate (1X) to each well and cover the plate with the Plate Sealer. Incubate 4-5 h at 37 o C.

4 User Protocol CBA042 Rev. 26 January 2006 JSW Page 4 of 5 Note: seal the plate tightly to prevent evaporation. Any changes in the reaction volume can influence the fluorescence reading. 7. Read and record the fluorescence measured at an excitation wavelength of ~324 nm and an emission wavelength of ~405 nm. Protocol Modifications for Inhibitor Screening 1. Following the wash cycles in step 5 add 100 µl Test Inhibitor (specifically prepared for inhibitor screening) to designated wells and cover the plate with the Plate Sealer. Incubate the plate for the desired amount of time at room temperature with gentle shaking. 2. In place of 100 µl Substrate (1X), add 20 µl Diluted Substrate (specifically diluted for inhibitor screening) to each well and gently shake the plate to mix. Incubate 4-5 h at 37 o C. Proceed to step 7. Calculation of Results 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. Assay Characteristics and Examples Assay Range ng/ml as measured with purified recombinant human TACE (Figure 1) rh TACE RFU R 2 = TACE [ng/ml] Figure 1: Activity of recombinant human TACE. The activity of increasing concentrations human recombinant TACE (Cat. No. PF133) was measured according to the Detailed Protocol above.

5 User Protocol CBA042 Rev. 26 January 2006 JSW Page 5 of 5 Assay Example 5000 TACE activity in cell lysates per mg protein 4000 RFU T98G DLD1 HT29 Figure 2: Activity of TACE/ADAM17 in biological samples: Human colorectal adenocarcinoma cells, DLD1 and HT-29 and human glioblastoma cells, T98G, were cultured in DMEM medium supplemented with 10% FCS and harvested at 80-90% confluency. Total cell lysates were prepared using CytoBuster TM Protein Extraction Reagent (Cat. No ) and the manufacturer s recommended protocol. TACE activity was measured according to the Detailed Protocol above. RFU is reported per mg protein. References Kirkegaard, T., et al Clin. Exp. Immunol. 135, 146. Neumann, U., et al Anal. Biochem. 328, 166. Skovronsky, D.M., et al J. Neurobiol., 49, 40. Black, R.A., et al Nature 385, 729. Moss, M.L., et al Nature 385, 733. Trademarks Calbiochem is a registered trademark of EMD Biosciences, Inc. CytoBuster and InnoZyme TM are trademarks of EMD Biosciences, Inc. Prices and availability are subject to change. Copyright 2005 EMD Biosciences, Inc., an affiliate of Merck KGaA, Darmstadt,. All rights reserved. Each product is sold with a limited warranty which is provided with each purchase. Each product is intended to be used for research purposes only. It is not to be used for drug or diagnostic purposes nor is it intended for human use. EMD Biosciences products may not be resold, modified for resale, or used to manufacture commercial products without written approval of EMD Biosciences.