207007 | Calcineurin Cellular Activity Assay Kit, Colorimetric

207007
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      Overview

      Replacement Information

      Key Specifications Table

      Species ReactivityDetection Methods
      H, M, R Colorimetric

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      207007-1KTT
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          Description
          OverviewA colorimetric assay kit for measuring cellular calcineurin (PP2B) enzyme activity. Assays are performed in a convenient 96-well plate format with all reagents necessary for measuring calcineurin activity. The RII Phosphopeptide Substrate (Cat. No. 207008), supplied with this kit, is the most efficient and selective substrate known for calcineurin. The free phosphate released is detected using malachite green.
          Catalogue Number207007
          Brand Family Calbiochem®
          SynonymsPP2B Assay Kit, Colorimetric
          Application Data
          Materials Required but Not Delivered Plate reader capable of measuring A620 to ≥3-decimal accuracy.
          Centrifuge capable of 100,000 x g RCF.
          Swing bucket centrifuge capable of 800 x g RCF.
          Pipettor capable of pipetting 5-100 µl accurately.
          Multi-channel pipettor capable of pipetting 100 µl (optional).
          Ice bucket to keep reagents cold until use.
          16 gauge needle/syringe.
          TBS buffer, 100 ml (150 mM NaCl, 20 mM Tris, pH 7.2)
          15 ml conical centrifuge tubes
          Biological test materials (e.g., tissue, cells)
          References
          ReferencesMondragon, A., et al. 1997. Biochemistry 36, 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 methodColorimetric
          Form96 Tests
          Format96-well plate
          Kit containsHuman Recombinant Calcineurin as a Positive Control, Bovine Brain Calmodulin, Calcineurin Substrate (Cat. No. 207008), Assay Buffer, EGTA Buffer, Lysis Buffer, Protease Inhibitor Cocktail, Phosphate Detection Agent, Phosphate Standard, Okadaic Acid (Cat. No. 495604), Desalting Column Resin, Chromatography Column, ½ volume 96-Well Plate, and a user protocol.
          Applications
          Biological Information
          Assay time7 h
          Sample TypeTissue/cellular extracts
          Species Reactivity
          • Human
          • Mouse
          • Rat
          Physicochemical Information
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          R PhraseR: 21/22-36/38

          Harmful in contact with skin and if swallowed.
          Irritating to eyes and skin.
          S PhraseS: 26-24/25

          In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
          Avoid contact with skin and eyes.
          Product Usage Statements
          Intended useThe Calbiochem® Calcineurin Cellular Activity Assay Kit is a complete colorimetric assay kit for measuring cellular calcineurin (PP-2B) phosphatase activity. It employs a convenient 96-well plate format with all reagents necessary for measuring calcineurin phosphatase activity in tissue/cellular extracts. Human recombinant calcineurin is included as a positive control. 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 malachite green assay and offers the following advantages: non-radioactive, convenient one-step detection, and excellent sensitivity.
          Storage and Shipping Information
          Ship Code Dry Ice Only
          Toxicity Multiple Toxicity Values, refer to MSDS
          Storage Multiple storage requirements
          Storage ConditionsStore all components except the plate (room temperature) at -70°C for the highest stability. The calcineurin enzyme component must be handled particularly carefully in order to retain maximal enzymatic activity. Thaw it quickly in a room temperature water bath or by rubbing between your fingers, then immediately store on ice. The remaining unused enzyme should be quickly refrozen by placing at -70°C. To minimize the number of freeze/thaw cycles, aliquot the calcineurin into separate tubes and store at -70°C.
          Avoid freeze/thaw Avoid freeze/thaw
          Do not freeze Ok to freeze
          Packaging Information
          Transport Information
          Supplemental Information
          Kit containsHuman Recombinant Calcineurin as a Positive Control, Bovine Brain Calmodulin, Calcineurin Substrate (Cat. No. 207008), Assay Buffer, EGTA Buffer, Lysis Buffer, Protease Inhibitor Cocktail, Phosphate Detection Agent, Phosphate Standard, Okadaic Acid (Cat. No. 495604), Desalting Column Resin, Chromatography Column, ½ volume 96-Well Plate, and a user protocol.
          Specifications

          Documentation

          SDS

          Title

          Safety Data Sheet (SDS) 

          Certificates of Analysis

          TitleLot Number
          207007

          References

          Reference overview
          Mondragon, A., et al. 1997. Biochemistry 36, 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.

          Brochure

          Title
          Protein Phosphatases Technical Bulletin
          User Protocol

          Revision13-November-2012 JSW
          SynonymsPP2B Assay Kit, Colorimetric
          Form96 Tests
          Format96-well plate
          Detection methodColorimetric
          Specieshuman, mouse, rat
          StorageStore all components except the plate (room temperature) at -70°C for the highest stability. The calcineurin enzyme component must be handled particularly carefully in order to retain maximal enzymatic activity. Thaw it quickly in a room temperature water bath or by rubbing between your fingers, then immediately store on ice. The remaining unused enzyme should be quickly refrozen by placing at -70°C. To minimize the number of freeze/thaw cycles, aliquot the calcineurin into separate tubes and store at -70°C.
          Intended useThe Calbiochem® Calcineurin Cellular Activity Assay Kit is a complete colorimetric assay kit for measuring cellular calcineurin (PP-2B) phosphatase activity. It employs a convenient 96-well plate format with all reagents necessary for measuring calcineurin phosphatase activity in tissue/cellular extracts. Human recombinant calcineurin is included as a positive control. 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 malachite green assay and offers the following advantages: non-radioactive, convenient one-step detection, and excellent sensitivity.
          BackgroundCalcineurin (CaN) is a neuronal form of the widely distributed Ca2+/calmodulin-dependent Ser/Thr protein phosphatase 2B (PP-2B). CaN is a heterodimer consisting of a catalytic A subunit (57-61 kDa) and a regulatory B subunit (19 kDa). The catalytic A subunit is composed of four functional domains: the catalytic core with sequence homology to PP-1 and PP-2A; binding sites for both calmodulin and CaN-regulatory subunit, and a C-terminal autoinhibitory domain.
          Materials provided• Calcineurin, Human, Recombinant (Kit Component No. KP8301): 500 U, Activity: 50 U/µl in 1X assay buffer. One unit is equal to 1 pmol per minute at 30°C
          • Calmodulin, Human, Recombinant (Kit Component No. KP8302): 100 µl, Supplied in 25 mM in H₂O.
          • RII Phosphopeptide (Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-pSer-Val-Ala-Ala-Glu) (Kit Component No. KP8313): 1 X 1.5 mg, The vial contains 1.5 mg net peptide
          • 2X Assay Buffer (Kit Component No. KP8303): 20 ml, Supplied as 200 mM NaCl, 100 mM Tris, 12 mM MgCl₂, 1 mM CaCl₂, 1 mM DTT, 0.05% NP-40, pH 7.5.
          • 2X EGTA Buffer (Kit Component No. KP8304): 1 ml, Supplied as 200 mM NaCl, 100 mM Tris, 20 mM EGTA, 12 mM MgCl₂, 1 mM DTT, 0.05% NP-40, pH 7.5.
          • Lysis Buffer (Kit Component No. KP8305): 1 x 40 ml, Supplied as 50 mM Tris, 1 mM DTT, 100 µM EDTA, 100 µM EGTA, 0.2% NP-40, pH 7.5.
          • Protease Inhibitor Cocktail (Kit Component No. KP8306): 2 tablets
          • GREEN™ Reagent (Kit Component No. KP8307): 20 ml
          • Phosphate Standard (Kit Component No. KP8308): 500 µl, Supplied as 80 µM in distilled H₂O.
          • Okadaic Acid (Kit Component No. KP8309): 325 µl, Supplied as 5 µM in 2X assay buffer. WARNING! LD₅₀ of < 200 mg/kg. May be carcinogenic/teratogenic.
          • Desalting Column and Resin (Kit Component No. KP8310): 5 ml, disposable polypropylene column and 1 g PG DG desalting resin. Store at room temperature; after rehydration store at 4°C.
          • 1/2 Volume Plate (Kit Component No. KP8312): 2 plates
          Materials Required but not provided Plate reader capable of measuring A620 to ≥3-decimal accuracy.
          Centrifuge capable of 100,000 x g RCF.
          Swing bucket centrifuge capable of 800 x g RCF.
          Pipettor capable of pipetting 5-100 µl accurately.
          Multi-channel pipettor capable of pipetting 100 µl (optional).
          Ice bucket to keep reagents cold until use.
          16 gauge needle/syringe.
          TBS buffer, 100 ml (150 mM NaCl, 20 mM Tris, pH 7.2)
          15 ml conical centrifuge tubes
          Biological test materials (e.g., tissue, cells)
          Precautions and recommendations• NOTE: Keep all samples in an ice bath until use, unless otherwise noted.

          The GREEN™ reagent is a highly sensitive phosphate detection solution. Free phosphate present on labware and in reagent solutions will greatly increase the background absorbance of the assay. This is detected visually as a change in color from yellow to green. Detergents used to clean labware may contain high levels of phosphate. Use caution by either rinsing labware with distilled H2O or employ unused plasticware. Do not use phosphate buffered saline (PBS) for any tissue/cell rinses, instead use TBS (Tris-buffered saline).
          PreparationTo prepare a tissue/cell extract for calcineurin activity assay: NOTE ON BIOLOGICAL SAMPLE MATERIAL: The following procedures have been tested for rat and mouse brain tissue. Other tissue or cell culture samples employed may require adjustment of this protocol for satisfactory results. 1. Add protease inhibitor tablets to lysis buffer immediately before use (1 tablet per 10 ml buffer). Vortex. 2. Obtain tissue, if fresh, excise quickly. 3. Rinse tissue quickly in ice-cold TBS and shake off/blot excess wetness. 4. Weigh the tissue in a centrifuge tube. 5. Add lysis buffer with protease inhibitor to tissue. Use 0.33-0.5 ml per gram of tissue. 6. Loosely break up cells by passing them through a 16 gauge needle. Avoid air bubbles. 7. Sediment at 100,000-200,000 x g in a centrifuge at 4°C for 45 min. Save the supernatant and label as "HSS" (high speed supernatant). 8. Freeze immediately at -70°C
          Reagent preparationRemoval of Free Phosphate from Extracts: To desalt tissue samples by gel filtration: NOTE: This procedure is intended to remove excess phosphate and nucleotides (which are slowly hydrolyzed to release free phosphate in the presence of the GREEN™ reagent) in the high speed supernatant (HSS) extract. Each 5 ml column can be used to de-salt several samples (3-6); it is important that the column be washed thoroughly between samples with phosphate-free H2O to remove excess phosphate from the column. 1. Re-hydrate desalting column resin in a 50 ml conical tube by adding 20 ml of phosphate-free distilled H2O. Vortex briefly. Allow to set for 4 h at room temperature or overnight at 4°C. 2. Decant the distilled H2O carefully. Then add fresh distilled H2O at a 1:1 ratio to the re-hydrated resin (~10 ml). 3. Add re-hydrated resin to the chromatography column to obtain a 5 ml settled-bed volume (~5.5 cm bed height). Remove the tip from the column and allow the H2O to drain by gravity. 4. Equilibrate the column by adding 8 ml of lysis buffer and allow to drain by gravity. 5. Place column in a 15 ml centrifuge tube. Centrifuge at 800 x g for 3 min at 4°C to displace column buffer. Discard flow-through buffer. 6. Place column in a clean 15 ml centrifuge tube. 7. Add up to 350 µl HSS sample, as prepared previously, to the column. 8. Centrifuge at 800 x g for 3 min. Save extract flow-through. This is the desalted cell lysate material to be tested for calcineurin activity, as outlined below. 9. Freeze sample immediately at -70°C. NOTE: The effective removal of phosphate/nucleotides from the extract should be tested qualitatively by adding 100 µl GREEN™ reagent to 1 µl extract and a separate sample of 1 µl distilled H2O. If no phosphate/nucleotides are present, both samples should remain yellow in color over a time period of 30 min at room temperature. The development of a visible green color indicates phosphate contamination, which must be eliminated from the samples before proceeding further. The resin can be re-used by rinsing with phosphate free water between samples. The flow-through can be tested for phosphate by adding green reagent. The flow-through will remain yellow when all phosphates have been removed from the column.
          Detailed protocolTo prepare the reagents for the assay:
          1. Thaw all kit components on ice, except for the GREEN™ reagent, which is can be thawed 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 assay well). For example, add 20 µl to 980 µl 2X assay buffer.
          3. Reconstitute substrate (RII phosphopeptide) with distilled H2O to 915 µl per 1.5 mg vial. This would yield a final peptide concentration of 1.64 mg/ml or 750 µM (10 µl are needed per assay well). Dispense unused substrate into aliquots and store at -70°C. Avoid freeze/thaw cycles.

          To prepare phosphate standard curve sample wells:
          1. Prepare 1 ml of 1X assay buffer (dilute 500 µl of 2X assay buffer with 500 µl of dH2O)
          2. 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 assay buffer (2X) to wells A1, and A2 (2 nmol PO4 standards)
          b. Add 50 µl of 1X assay buffer (prepared in step 1 above) to wells B1-H1 and wells B2 to H2. (remaining standard concentrations)
          c. Add 50 µl of 80 µM phosphate standard wells A1 and A2 of assay plate. Mix thoroughly by pippetting up and down several times..
          d. Remove 50 µl from well A1 and add it to well B1. Mix thoroughly by pippetting 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 fo the wells in column 2 (standard curve duplicates)

          To prepare the calcineurin activity assay sample wells: (See tables 1 and 2)

          Background (no substrate):
          (Control for background phosphate/interfering substances)

          1. Add 20 µl distilled H2O to appropriate wells.
          2. Add 25 µl 2X assay buffer with calmodulin to each well.

          Total phosphatase activity wells:
          (Total phosphatase activity in the extract)

          1. Add 10 µl dH2O to each well.
          2. Add 25 µl 2X assay buffer with calmodulin to each well.

          EGTA buffer (Ca2+/CaM Free):
          [Toal activity less PP-2B (Calcineurin)]

          1. Add 10 µl dH2O to each well.
          2. Add 25 µl 2X EGTA buffer to each well.

          Okadaic acid:
          (Total activity less PP-1 and PP-2A)

          1. Add 5 µl dH2O to each well.
          2. Add 25 µl 2X assay buffer with calmodulin to each well.
          3. Add 5 µl okadaic acid

          Okadaic acid + EGTA
          (Total activity less PP-1, PP-2A, and PP-2B)

          1. Add 5 µl dH2O to each well.
          2. Add 25 µl 2X EGTA buffer to each well.
          3. Add 5 µl okadaic acid.

          Positive control (calcineurin enzyme)
          (Purified CaN enzyme positive control)

          1. Add 10 µl dH2O to each well.
          2. Add 25 µl 2X assay buffer with calmodulin to each well.

          Add Phosphopeptide Substrate:
          3. Add 10 µl phosphopeptide to each well of the calcineurin samples except the background control. DO NOT ADD SUBSTRATE TO THE PHOSPHATE STANDARD CURVE SAMPLES.
          4. Equilibrate plate to reaction temperature (e.g., 30°C) for 10 min.

          To initiate the calcineurin assay:
          5. Add 5 µl extract or calcineurin (dilute to 8U/µl with 1x Assay Buffer just prior to use) to appropriate wells. For sample extract wells, it may be necessary to dilute the HSS tissue extract (e.g., 1:5-1:10 in lysis buffer). For calcineurin positive control, add 5 µl of calcineurin enzyme (40 U/well).
          6. Incubate the plate at reaction temperature for desired duration (e.g., 30 min at 30°C).

          To terminate the reactions:
          7. After incubating wells for desired duration, terminate reactions by adding 100 µl GREEN™ reagent to ALL samples including the phosphate standard curve.
          8. Allow color to develop 20-30 min (the color will change from yellow to green), making sure all wells spend approximately the same time with the reagent before reading on microplate reader.
          9. Read A620 on plate reader.
          10. Perform data analysis as indicated below.

          Table 1: Example of Plate Samples

          *For highest accuracy, perform all samples in duplicate.


          Table 2: Typical Assay Components

          a. Add cellular extract b. Add calcineurin enzyme

          CalculationsData Analysis Phosphate (PO4) standard curve: 1. Plot standard curve data as A620 versus nmol PO4 (see Figure 1). 2. Obtain a line-fit to the data using an appropriate routine 3. Use the slope and the Y-intercept to calculate amount of phosphate released for the experimental data. NOTE: For highest accuracy, a standard curve must be performed for each new set of assay data. This will normalize for variations in free phosphate in samples, time of incubation with the GREEN™ Reagent, and other experimental factors.

          Figure 1: Standard Curve

          Conversion of A620 to amount of phosphate released 1. Convert A620 data line into the amount of phosphate released using the standard curve line-fit data from above: Phosphate released = (A620-Yint)/slope Example: Standard Curve Slope = 0.3 A620/nmol phosphate Standard Curve Yint = 0.01 Sample A620 = 0.4 Phosphate released = (0.4-0.001)/0.3 = 1.33 nmol Data Reduction to Determine Calcineurin Phosphatase PRECAUTIONS: The procedures for data analysis that follows are intended only as a guideline. The individual user must determine the suitability of this analysis for their particular experimental protocol. Additional controls and other samples may be appropriate for accurate analysis. ANALYSIS DESCRIPTION: This assay uses RII phosphopeptide, the most well-known substrate for PP-2B. Nonetheless, in cellular extracts, the phospho-group is cleaved by other competing phosphatases. Thus, a series of conditions must be employed to discriminate between PP2B activity and that of other phosphatases. CaN requires calcium for its activity, thus the EGTA buffer sample represents total phosphatase activity less CaN. Okadaic Acid at 100 and 500 nM is known to completely inhibit PP-1 and PP-2A, while it has no effect on CaN (see Figure 3). Finally, okadaic acid + EGTA buffer inhibits PP-1, PP-2A, and PP-2B, but not PP-2C. Thus for a given biological system, the analysis of these samples allow for the quantification of calcineurin activity in a cellular extract. Additional experimental modulation of the cellular extracts may be desirable. Inhibitors of calcineurin, calmodulin, and calcium ion modulations may be appropriate. Calculation: 1. Subtract the background phosphate released from each sample except the positive control. 2. Plot a graph analogous to Figure 2. Use either A620 or phosphate released for the Y-axis. 3. Determine the contribution of calcineurin; eq. 1. CaN (PP-2B) = Total - EGTA Buffer or eq. 2. CaN (PP-2B) = OA - (OA + EGTA) Eq. 1 is a conventional method to report CaN activity. However, the user must determine the most appropriate analysis for their specific experimental goal.
          Example data

          Figure 2: Cellular Calcineurin (CaN) Assay using Mouse Brain

          Phosphatase activity from freshly prepared mouse brain extract. Prior to gel filtration, the extract was diluted 1:1 in lysis buffer containing protease inhibitors. After gel filtration and prior to the phosphatase assay, the extract was diluted 1/25 in lysis buffer. Well contents were as in Table 2. The reaction was incubated for 30 min at 30°C.

          Figure 3: Calcineurin (CaN) Positive Control

          The CaN was incubated 1 h at 30°C under various buffer conditions. The results demonstrate that CaN activity is inhibited by the EGTA buffer, but not inhibited by 100 nM or 500 nM concentrations of okadaic acid.

          Registered TrademarksCalbiochem® is a registered trademark of Merck, KGaA, Darmstadt.
          Interactive Pathways™ is a trademark of Merck, KGaA, Darmstadt.
          GREEN™ Reagent is a trademark of Biomol International