Certificate of Analysis

Certificate of Analysis 10 Old Barn Road Lake Placid, NY 12946 Technical Support: T: 800 548-7853 F: 518 523-4513 email: techserv@upstate.com Sales Department: T: 800 233-3991 F: 781 890-7738 Licensing Dept.: 800 310-4659 www.upstate.com Alkaline/Acid Phosphatase Assay Kit (R-R-A-pS-V-A) Kit Components Malachite Green (Solution A), Catalog # 20-105. One vial containing 20ml of 0.034% malachite green in 10mM ammonium molybdate, 1N HCl and 3.4% ethanol. Malachite Green Additive (Solution B), Catalog # 20-104. One vial containing 0.5ml of 1% Tween 20. Phosphate Standard (Solution C), Catalog # 20-103. One vial containing 1ml (1.0mM) of 1mM potassium phosphate monobasic, KH 2 PO 4. Serine Phosphopeptide (RRApSVA), Catalog # 12-220, Lot # 26616. One vial containing 1mg lyophilized powder. p-nitrophenyl Phosphate (pnpp), Catalog # 20-106. One vial containing 2 tablets, each tablet contains 5mg pnpp. NiCl 2, 40mM, Catalog # 20-178. One vial containing 1ml of 40mM NiCl 2. pnpp Ser/Thr Assay Buffer, Catalog # 20-179. One vial containing 20ml of 50mM Tris-HCl, ph 7.0, 0.1mM CaCl 2. 96-Well Microtiter Plate (½ volume flat bottom plate). Kit Description Quantity: 100 assays per kit. Quality Control: The components of this kit have matched to optimize sensitivity and range of detection. Phosphate released is determined by comparing absorbance to standard curve of step 2. Using this kit, 0.5U of calf intestine alkaline phosphatase released 66.9pmol phosphate per minute using the phosphopeptide (RRApSVA). Storage and Stability: Stable for 1 year at 4 C from date of shipment. Use: Read the enclosed protocol before use. Do not perform the Malachite Green Assay using phosphate buffers or with biological samples that contain high levels of free phosphate. Remove free phosphate from samples by desalting column or dialysis. References: 1. Harder, K.W., et al., Biochem. J. 298: 395-401, 1994. 2. Donella-Deana, A., et al., Biochim. Biophys. Acta 1094: 1309, 1991. FOR RESEARCH USE ONLY. NOT RECOMMENDED OR INTENDED FOR DIAGNOSIS OF DISEASE IN HUMANS OR ANIMALS DO NOT USE IN HUMANS

Page Two of Five Malachite Green Phosphatase Assay 1. Preparation of Malachite Green Phosphate Detection Solution Add 10µl of Solution B to each 1ml of Solution A. Mix only enough reagent as required for use that day. The solution may be kept at room temperature during use. 2. Phosphate Standard Curve Dilute 100µl of Catalog # 20-103, Phosphate Standard (Solution C) with 900µl of distilled water to make 0.1mM working solution. Use this solution to prepare a phosphate standard curve as described in the table below. The standard curve should range between 200 and 2000pmoles per well. Volume of diluted stock (0.1mM) Volume of distilled water or diluent Picomoles of Phosphate per 25µl 200µl 180 160 140 120 100 80 60 40 20 0 50µl 70 90 110 130 150 170 190 210 230 250 2000 1800 1600 1400 1200 1000 800 600 400 200 0 a) Transfer 25µl of each phosphate standard to wells of a microtiter plate. Use distilled water as a blank. b) Add 100µl of Malachite Green Solution prepared as described in Step 1. Mix carefully without creating bubbles (bubbles interfere with measurement of absorbance). c) Allow color development to proceed for 15 minutes at room temperature. d) Measure absorbance at a wavelength between 620 and 660nm in a microtiter plate reader. Subtract the absorbance of blank solution from standards. The absorbance readings are slightly lower if the assay is read at 650-660nm as compared to 620nm. e) Plot pmoles phosphate vs. absorbance for the standard curve. See a representative standard curve below. Phosphate Standard Curve 1 0.8 0.6 0.4 0.2 0 0 200 400 600 800 1000 1200 1400 1600 1800 2000 pmoles of phosphate

Page Three of Five 3. Phosphopeptide Preparation a) Dissolve 1mg peptide (MW 781Da) in 1285µl of sterile, distilled water to prepare a 1mM solution. b) Aliquot peptide solution and store at -20 C as necessary. If the Malachite Green Phosphatase Assay is used primarily for kinetic analysis of purified Ser-Thr phosphatases, use the table below to prepare dilutions for velocity versus substrate reactions. The Kit provides enough reagents for approximately 18 sets of velocity vs. substrate reactions. Final [peptide]/ reaction 750µM 500µM 350µM 250µM 175µM 100µM Volume peptide/25µl assay 20.0µl 12.5µl 8.8µl 6.3µl 4.5µl 2.5µl 4. Enzyme Assay This kit is designed for assaying phosphatase activity directly in the 96-well microtiter plate. Enzyme reactions are performed in a final volume of 25-50µl (use the same volume as that used for the standard curve). Before proceeding, check that your enzyme preparation/samples are free of contaminating phosphate. Determine phosphate contamination by adding Malachite Green Detection Solution (100µl), step 1 above, to 2-5µl of enzyme preparation or experimental sample. Any samples contaminated with phosphate will turn green. Remove phosphate by desalting column or dialysis. Read absorbance at 620-660nm and compare to standard curve to determine level of any contaminating phosphate. Stock solutions: Enzyme Assay for Alkaline Phosphatases by Malachite Green Detection System A. pnpp Ser/Thr Assay Buffer: 50mM Tris-HCl, 0.1mM CaCl 2, ph 7.0. B. NiCl 2 : 40mM NiCl 2 in sterile, distilled water. C. BSA Solution: 5mg/ml. D. Serine Phosphopeptide: Dilute to 1mM with 1285µl of sterile, distilled water. See Step 3a above. E. Malachite Green Solution: See Step 1, page two. F. Enzyme: 0.1-0.5 Units per well. 1. Mix in one microtiter well: 5µl NiCl 2, 5µl BSA, 5µl phosphopeptide stock solution and 0.1-0.5 Units of enzyme. 2. Adjust volume to 80µl with pnpp Ser/Thr Assay Buffer. 3. Transfer 25µl aliquots to new microtiter wells; test in triplicate. Test samples versus positive control. Prepare a blank the same as the sample, but without enzyme (replace enzyme with an equal volume of pnpp Ser/Thr Assay Buffer). 4. Incubate the reaction for 10-15 minutes at 37 C. This step greatly speeds up the reaction. 5. Detect phosphatase activity by addition of 100µl Malachite Green solution. 6. Incubate for 15-20 minutes at room temperature. 7. The assay is read against blank at 650nm (620nm to 660nm). 8. Compare absorbance value to standard curve to calculate phosphatase activity.

Page Four of Five Stock Solutions: Enzyme Assay for Acid Phosphatase by Malachite Green Detection A. Acid Assay Buffer: 0.1M sodium acetate, ph 6.0, 0.2% Triton X-100, 1mM EDTA. B. Serine Phosphopeptide: Dilute to 1mM with 1285µl of sterile, distilled water. See Step 3a, page three. C. Malachite Green Solution. See Step 1, page two. D. Enzyme: 0.1-0.5 Units per well. 1. Dilute test acid phosphatase and positive control acid phosphatase with acid assay buffer to a final concentration of 0.02 Units/20µl. 2. Add 20µl of acid phosphatase solution to microtiter plate. 3. Add 5µl of the 1mM phosphopeptide stock solution. Final concentration = 200µM. 4. Incubate 10-20 minutes at 37 C. Incubation time depends on quantity and specific activity of enzyme preparation. 5. Add 100µl of Malachite Green solution and incubate for 15-30 minutes. 6. Read absorbance at 650nm (620nm-660nm) against blank. 7. Compare absorbance value to standard curve to calculate phosphatase activity. Stock Solutions: Microtiter Assay of Protein Phosphatases by pnpp Hydrolysis A. pnpp Ser/Thr Assay Buffer: 50mM Tris-HCl, 0.1mM CaCl 2, ph 7.0. B. NiCl 2 : 40mM NiCl 2 in distilled water. C. BSA Solution: 5mg/ml. D. Substrate: 1.5mg/ml pnpp in 50mM Tris-HCl, ph 7.0, freshly prepared before assay. E. Enzyme: 0.1-0.5 Units per well. F. Stopping Solution (optional): 13% K 2 HPO 4 ; the stopping solution reduces the intensity of the phosphatase reaction. 1. Mix in one microtiter well: 5µl NiCl 2, 5µl BSA and 0.1-0.5 Units of enzyme. 2. Adjust volume to 80µl with pnpp Ser/Thr Assay Buffer. 3. Transfer 25µl aliquots to new microtiter wells; test in triplicate. Test samples versus positive control. Prepare a blank the same as the sample, but without enzyme (replace enzyme with an equal volume of pnpp Ser/Thr Assay Buffer). 4. Incubate the reaction mixture for 15-30 minutes at 37 C. This step greatly speeds up the reaction. 5. Add 120µl of substrate to start the phosphatase reaction. 6. Incubate for 30-45 minutes at 37 C to allow for color development. 7. The reaction is stopped by adding 20µl of stopping solution (optional). 8. The assay is read against the blank at 405-410nm.

Page Five of Five The A 405 reflects phosphatase activity. One Unit of activity is equivalent to 1nmol pnpp hydrolyzed per minute. To determine phosphatase activity, use the following equation: (Sample volume in liters) x A 405 (ε) x (Path length of light in cm*) x (Assay time in minutes) x (units or µg of enzyme) **Multiply the answer to this equation by 10 9 to convert the result from moles/min/u (or moles/min/µg) to nmol/min/u (or nmol/min/µg)** ε = Extinction coefficient for pnpp at A 405 = 1.78 x 10 4 M -1 cm -1 *Pathlength of light will vary, depending on type of microtiter plate or cuvette used.