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IRAK2, Active IRAK2, Active Catalogue # Aliquot Size I10-10BG-05 5 µg I10-10BG-10 10 µg I10-10BG-20 20 µg IRAK2, Active Full-length recombinant protein expressed in Sf9 cells Catalog # I10-10BG Lot # V117 -2B Product Description Specific Activity Recombinant full-length human IRAK2 was expressed by 280,000 baculovirus in Sf9 insect cells using an N-terminal GST tag. The gene accession number is NM_001570 . 210,000 Gene Aliases 140,000 IRAK-2, MGC150550 70,000 (RLU) Activity Formulation 0 0 200 400 600 800 Recombinant protein stored in 50mM Tris-HCl, pH 7.5, Protein (ng) 150mM NaCl, 10mM glutathione, 0.1mM EDTA, 0.25mM DTT, 0.1mM PMSF, 25% glycerol. The specific activity of IRAK2 was determined to be 6 nmol /min/mg as per activity assay protocol. Storage and Stability Purity o Store product at –70 C. For optimal storage, aliquot target into smaller quantities after centrifugation and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles. The purity of IRAK2 was determined to be >75% by densitometry, approx. MW 103kDa . Scientific Background Interleukin-1 receptor-associated kinase 2 (IRAK2) is important downstream signaling components of Toll-like receptors (TLRs) (1). IRAKs were first described as signal transducers for IL-1 and later have been implicated in signal transduction of other members of the Toll/IL-1 IRAK2, Active receptor family. The interleukin-1 receptor (IL-1R) signaling Full-length recombinant protein expressed in Sf9 cells pathway leads to NF κB activation in mammals. To date, Catalog Number I10-10BG four mammalian IRAKs have been identified (IRAK-1, Specific Activity 6 nmol/min/mg IRAK-2, IRAK-4, and IRAK-M) (2). It is believed that IRAK-2 Specific Lot Number could be an additional therapeutic target for inhibiting IL- V117-2B Purity >75% 1-induced inflammation. Format 1µg in 10µl Concentration 0.1 µ g/ µl References Stability 1yr at –70 oC from date of shipment o Storage & Shipping Store product at –70 C. For optimal 1. Muzio, M. et al: IRAK (Pelle) family member IRAK-2 and storage, aliquot target into smaller quantities after centrifugation and MyD88 as proximal mediators of IL-1 signaling. Science. store at recommended temperature. 1997;278:1612-5. For most favorable performance, 2. Wesche, H. et al: IRAK-M is a novel member of the avoid repeated handling and multiple Pelle/interleukin-1 receptor-associated kinase (IRAK) family. freeze/thaw cycles. Product shipped J. Biol. Chem. 1999; 274:19403-10. on dry ice. To place your order, please contact us by phone 1-(604)-232-4600, fax 1-604-232-4601 or by email: [email protected] www.signalchem.com FOR IN VITRO RESEARCH PURPOSES ONLY. NOT INTENDED FOR USE IN HUMAN OR ANIMALS. ADP-Glo TM Activity Assay Protocol Reaction Components Active Kinase (Catalog #: I10-10G ) ADP-Glo TM Kinase Assay Kit (Promega, Cat # V9101) Active IRAK2 (0.1 µg/ µl) diluted with Kinase Dilution Buffer III (Catalog #: K23-09 ) and assayed as outlined in ADP solution, 10 mM sample activity plot. (Note: these are suggested working ADP-Glo TM Reagent dilutions and it is recommended that the researcher Kinase Detection Reagent perform a serial dilution of Active IRAK2 for optimal results). Kinase Dilution Buffer III (Catalog #: K23 -09 ) 250 µµµM ATP Assay Solution Kinase Assay Buffer I (Catalog #: K01-09 ) diluted at a Prepare ATP assay solution by dissolving 0.55mg of ATP in 4ml 1:4 ratio (5X dilution) with distilled H2O. of Kinase Assay Buffer I (Catalog #: K01-09 ). Store 200 µl aliquots at –20 oC. Kinase Assay Buffe r I (Catalog #: K01 -09 ) Substrate (Catalog #: A05-58B) Buffer components: 25mM MOPS, pH 7. 2, 12.5mM β- Modified AKT Substrate peptide (modified-CKRPRAASFAE) glycerol-phosphate, 25mM MgC1 2, 5mM EGTA, 2mM was diluted in distilled H2O to a final concentration of EDTA. Add 0.25mM DTT to Kinase Assay Buffer prior to 1mg/ml. use. Assay Protocol The IRAK2 assay is performed using the ADP-Glo TM Kinase Assay kit (Promega; Cat# V9101) which quantifies the amount of ADP produced by the IRAK2 reaction. The ADP- Glo TM Reagent is added to terminate the kinase reaction and to deplete the remaining ATP, and then the Kinase Detection Reagent is added to convert ADP to ATP and to measure the newly synthesized ATP using luciferase/luciferin reaction. Step 1. Thaw the Active IRAK2, Kinase Assay Buffer, Substrate and Kinase Dilution Buffer on ice. Step 2. In a pre-cooled 96-well opaque plate, add the following reaction components bringing the initial reaction volume up to 20 µl: Component 1. 10 µl of diluted Active IRAK2 (Catalog #I10-10BG) Component 2. 5µl of 1mg/ml stock solution of substrate (Catalog #A05-58B) Component 3. 5µl Kinase Dilution Buffer III Step 3. Set up the blank control as outlined in step 3, excluding the addition of the substrate. Replace the substrate with an equal volume of Kinase Dilution Buffer III. Step 4. Initiate the reaction by the addition of 5 µl of 250 µM ATP Assay Solution thereby bringing the final volume up to 25 µl. incubate the mixture in a water bath at 30 oC for 15 minutes. Step 5. After the 15 minute incubation period, terminate the reaction and deplete the remaining ATP by adding 25 µl of ADP-Glo TM Reagent. Shake the 96-well plate and then incubate the reaction mixture for another 40 minute at ambient temperature. Step 6. Then add 50 µl of the Kinase Detection Reagent to the 96-well plate and incubate the reaction mixture for another 30 minute at ambient temperature. Step 7. Read the 96-well reaction plate using the KinaseGlo Luminescence Protocol on a GloMax plate reader (Promega; Cat# E7031). Step 8. Determine the corrected activity (RLU) by removing the blank control value (see Step 3) for each sample and calculate the kinase specific activity as outlined below. Calculation of Specific Activity of ADP (RLU/pmol) From ADP standard curve, determine RLU/pmol of ADP Kinase Specific Activity (SA) (pmol/min/ µµµg or nmol/min/mg) Corrected RLU from reaction / [(SA of ADP in RLU/pmol)*(Reaction time in min)*(Enzyme amount in µg or mg) To place your order, please contact us by phone 1-(604)-232-4600, fax 1-604-232-4601 or by email: [email protected] www.signalchem.com FOR IN VITRO RESEARCH PURPOSES ONLY. NOT INTENDED FOR USE IN HUMAN OR ANIMALS. .
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