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A colorimetric assay kit for measuring calcineurin (PP2B) activity and for inhibitor screening. The assay is performed using RII Phosphopeptide Substrate (Cat. No. 207008).
Catalogue Number
207005
Brand Family
Calbiochem®
Synonyms
PP2B Assay Kit, Colorimetric
Materials Required but Not Delivered
• Plate reader capable of measuring A620 to ≥3-decimal accuracy. • Pipette capable of pipetting 5-100 µl accurately. • Multi-channel pipette capable of pipetting 100 µl (optional). • Ice bucket to keep reagents cold until use.
References
References
Mondragon, A. et al. 1997. Biochemistry36, 4934. Donella-Deana, A., et al. 1994. Eur. J. Biochem. 219, 109. Enz, A., et al. 1994. Anal. Biochem. 216, 147. Harder, K.W., et al. 1994. Biochem. J. 298, 395. Martin, B., et al. 1985. J. Biol. Chem. 260, 14932.
Product Information
Detection method
Colorimetric
Form
96 Tests
Format
96-well plate
Kit contains
Human Recombinant Calcineurin, Bovine Brain Calmodulin, RII Phosphopeptide Substrate, Assay Buffer, Phosphate Detection Reagent, Phosphate Calibration Standard, ½ volume 96-Well Plate, and a user protocol.
The Calbiochem® Calcineurin Assay Kit is a complete colorimetric assay kit that is suitable for measuring calcineurin activity. It offers the following advantages: • NON-RADIOACTIVE • CONVENIENT 1-STEP DETECTION - no mixing • 96-WELL-PLATE FORMAT- for high-throughput applications.
Please Note: This kit has not been tested for any other phosphatase activity aside from calcineurin (PP2B) activity and inhibitor screening.
Storage and Shipping Information
Ship Code
Dry Ice Only
Toxicity
Multiple Toxicity Values, refer to MSDS
Storage
≤ -70°C
Storage Conditions
Store the 96-Well Plate at room temperature and all other components at -70°C for the highest stability. In particular, calcineurin must be handled with utmost care in order to retain its maximal enzymatic activity. Thaw it quickly in a water bath, maintained at room temperature, or by rubbing between fingers, then immediately store on an ice bath. Any remaining unused enzyme should be quickly refrozen by placing at -70°C. To minimize the number of freeze/thaw cycles, we recommend to aliquot the calcineurin into separate tubes and then store at -70°C.
Do not freeze
Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit contains
Human Recombinant Calcineurin, Bovine Brain Calmodulin, RII Phosphopeptide Substrate, Assay Buffer, Phosphate Detection Reagent, Phosphate Calibration Standard, ½ volume 96-Well Plate, and a user protocol.
Mondragon, A. et al. 1997. Biochemistry36, 4934. Donella-Deana, A., et al. 1994. Eur. J. Biochem. 219, 109. Enz, A., et al. 1994. Anal. Biochem. 216, 147. Harder, K.W., et al. 1994. Biochem. J. 298, 395. Martin, B., et al. 1985. J. Biol. Chem. 260, 14932.
Store the 96-Well Plate at room temperature and all other components at -70°C for the highest stability. In particular, calcineurin must be handled with utmost care in order to retain its maximal enzymatic activity. Thaw it quickly in a water bath, maintained at room temperature, or by rubbing between fingers, then immediately store on an ice bath. Any remaining unused enzyme should be quickly refrozen by placing at -70°C. To minimize the number of freeze/thaw cycles, we recommend to aliquot the calcineurin into separate tubes and then store at -70°C.
Intended use
The Calbiochem® Calcineurin Assay Kit is a complete colorimetric assay kit that is suitable for measuring calcineurin activity. It offers the following advantages: • NON-RADIOACTIVE • CONVENIENT 1-STEP DETECTION - no mixing • 96-WELL-PLATE FORMAT- for high-throughput applications.
Please Note: This kit has not been tested for any other phosphatase activity aside from calcineurin (PP2B) activity and inhibitor screening.
Background
Calcineurin (CaN), a Ca2+/calmodulin-regulated Ser/Thr protein phosphatase (PP-2B), plays a critical role in Ca2+-mediated signal transduction and T-cell activation. In the nervous system, it is mainly localized in neurons that are vulnerable to ischemia and traumatic insults. CaN is a heterodimer consisting of a catalytic subunit A (57-61 kDa) and a regulatory subunit B (19 kDa). The catalytic A subunit is composed of four functional domains: the catalytic core with sequence homology to PP1 and PP2A (located between residues 71-235 in the rat brain αδ isoform), binding sites for both calmodulin (residues 391-414) and CaN B-regulatory subunit, and a C-terminal (residues 457-482) autoinhibitory domain.
Principles of the assay
The Calbiochem® Calcineurin Assay Kit employs a convenient 96-well plate format with all reagents necessary for measuring calcineurin activity. The RII phosphopeptide substrate, supplied with this kit, is the most efficient and outstanding peptide substrate known for calcineurin. The detection of free phosphate released is based on the classic Malachite Green assay. This kit incorporates human calcineurin Aα (MW = 60 kDa) + calcineurin B (MW = 15 kDa) heterodimers expressed in an E. coli expression system. Both subunits were coexpressed in a construct with yeast myristoyl-CoA:protein N-myristoyl-transferase. The resulting highly active calcineurin is N-myristoylated on the B subunit, similar to the native protein.
Materials provided
• Calcineurin, Human Recombinant (Kit Component No. KP3501): 1 vial, 5000 Units at 100 U/µl in 1X Assay Buffer. 1 U = pmol/min at 30°C. • Calmodulin, Human Recombinant (Kit Component No. KP3502): 1 vial, 100 µl of 25 µM (in H₂O). • Substrate (RII phosphopeptide, sequence Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-pSer-Val-Ala-Ala-Glu; MW = 2192.0) (Kit Component No. KP3503): 1 vial, 1.5 MG, (1.5 mg net peptide/vial). • 2X Assay Buffer (Kit Component No. KP3504): 1 bottle, 20 ml, containing 200 mM NaCl, 100 mM Tris, 12 mM MgCl₂, 1 mM CaCl₂, 1 mM DTT, 0.05% NP-40, pH 7.5. • Malachite Green Reagent (Kit Component No. KP3505): 1 bottle, 20 ml. • Phosphate Standard (Kit Component No. KP3506): 0.5 ml of 80 µM solution in distilled H₂O. • Half Volume 96-Well Plate (Kit Component No. KP3507): 1.
Materials Required but not provided
• Plate reader capable of measuring A620 to ≥3-decimal accuracy. • Pipette capable of pipetting 5-100 µl accurately. • Multi-channel pipette capable of pipetting 100 µl (optional). • Ice bucket to keep reagents cold until use.
Detailed protocol
NOTE ON HANDLING: Keep all samples on ice bath until use, unless otherwise noted.
PRECAUTIONS: The Malachite Green reagent is a highly sensitive phosphate detection solution. Free phosphate present on labware and in reagent solutions will greatly increase the background absorbance. This is detected visually as a change in color from yellow to green. Use only phosphate-free detergents to clean labware. Rinse glassware in distilled water or employ unused plasticware.
To Prepare Reagents for the Assay: 1. Thaw all kit components and maintain calcineurin, calmodulin, and 2X Assay Buffer on an ice bath. Store Malachite Green reagent at room temperature. 2. Add calmodulin to the 2X assay buffer: Dilute calmodulin 1:50 in 2X assay buffer to the required quantity (25 µl are required per well). For example, add 20 µl to 980 µl of 2X assay buffer. 3. Reconstitute substrate (RII phosphopeptide) with distilled H2O to 0.75 mM (1.64 mg/ml): Add 915 µl distilled H2O to the 1.5 mg vial (10 µl are needed per assay well).
To Prepare a Standard Curve: 4. Prepare 1 ml of 1X assay buffer (dilute 500 µl of 2X assay buffer with 500 µl of dH2O) 5. Perform 1:1 serial dilutions of phosphate standard and an assay buffer blank. Concentrations of 40, 20, 10, 5, 2.5, 1.25, and 0.625 µM correspond to 2, 1, 0.5, 0.25, 0.125, 0.063, and 0.031 nmol PO4 (see Table 1): a) Add 50 µl of of 2X assay buffer (KI-128) to each wells A1, and A2 (2 nmol PO4 standards). b) Add 50 µl 1X assay buffer (prepared in step 4 above) to wells B1-H1 and wells B2-H2 (remaining standard concentrations) c) Add 50 µl of 80 µM phosphate standard to well A1 and A2 of assay plate. Mix thoroughly by pipetting up and down several times. d) Remove 50 µl from well A1 and add it to well B1. Mix thoroughly by pipetting up and down several times. e) Remove 50 µl from well B1 and add it to well C1. f) Mix thoroughly and repeat for wells D1-G1. At well G1, Remove 50 µl and discard. DO NOT PROCEED TO WELL H1 (assay buffer blank). Final volume=50 µl. g) Repeat serial dilution for the wells in column 2 (standard curve duplicates).
Determination of the Time Course/Linearity of Assay: 6. Add 25 µl of the 2X assay buffer with calmodulin (step 2) to wells designated for linearity assay (see Table 1). 7. Dilute calcineurin to 8 U/µl in 1X assay buffer and add 5 µl of calcineurin to wells. Final amount of calcineurin = 40 U per well. 8. Add 10 µl of distilled H2O to each well. 9. Designate a reaction time to each well (e.g.: 60 min, 40 min, 30 min, 20 min, 10 min, 5 min, 2 min, 0 min). 10. Equilibrate the plate to the desired reaction temperature (e.g., 30°C). 11. Start reaction by addition of 10 µl phosphopeptide substrate (0.75 mM from step 3) at appropriate time point. For convenience, carry out the additions in the reverse time order so that all incubations end at the same time (e.g., add 60 min time point at t = 0; add 5 min at t = 55 min, etc.). Final substrate concentration = 0.15 mM.
To Prepare a Test Sample/Inhibition Assay: 12. Add 25 µl of the assay buffer with calmodulin (step 2) to designated wells (Table 1). 13. Dilute calcineurin to 8 U/µl in 1X assay buffer and add 5 µl of calcineurin to wells. Final amount of calcineurin = 40 U per well. 14. Add 10 µl distilled H2O to control wells. 15. Add 10 µl of the test sample/inhibitor (dissolved in distilled H2O) to test sample wells. 16. Allow test sample/inhibitor to equilibrate to reaction temperature (e.g., 30°C) for 10 min. 17. Start reaction by addition of 10 µl phosphopeptide substrate (0.75 mM from step 3). Final concentration = 0.15 mM. Allow reaction to proceed for a time period in which the reaction is linear (~10 min, see below).
To Terminate Reactions: 18. After incubating wells for desired period of time, terminate reactions with 100 µl Malachite Green reagent. 19. Allow color to develop for 20-30 min. Allow the same incubation period for each well. 20. Read A620 on the plate reader. 21. Perform data analysis (see below).
NOTE: Save the 96-Well plate for future use. Use only the unused wells.
Calculations
Phosphate Standard Curve
1. Plot standard curve data as A620 versus nmol phosphate (see Figure 1). 2. Obtain a line-fit to the data using an appropriate routine. 3. Use the slope and Y-intercept to calculate amount of phosphate released for other experimental data (e.g., time course and experimental data). NOTE: For highest accuracy, prepare a standard curve each time the samples are run. This will normalize for any
variations in free phosphate in samples, incubation time with the Malachite Green reagent, and any other factors.
Conversion of A620 to Amount Phosphate Released 1. Convert A620 data into the amount of phosphate released using the standard curve line-fit data, from above: Phosphate released = (A620 - Yint)/slope EXAMPLE:
Time Course/Linearity Curve1. If the zero time (Table 1, well # H3,4) has a significant value, subtract this number from all samples. This is
background phosphate in the samples. 2. Plot A620 versus reaction time (see Figure 2). Alternatively, the A620 can be converted to phosphate released, as above.
3. Determine the reaction time range in which the amount of phosphate released is linear. In Figure 2, this range is from zero to 60 min. This value may vary depending on reaction conditions as well as storage/handling of
calcineurin. The duration of incubation can be lengthened by decreasing the amount of calcineurin in the assay and by lowering the assay temperature. For accurate results, it is important to perform inhibitor/agonist assays
under linear assay conditions.
Calmodulin Activation of Calcineurin Activity
Figure 3 illustrates the activation of calcineurin's phosphatase activity by calmodulin. In the presence (+) of calmodulin, calcineurin's activity is high. In the absence (-) of calmodulin, calcineurin activity is relatively low.
Registered Trademarks
Calbiochem® is a registered trademark of Merck KGaA, Darmstadt Interactive Pathways™ is a trademark of Merck KGaA, Darmstadt.
