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207008 | Calcineurin Substrate - Calbiochem

207008
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      Overview

      Replacement Information

      Pricing & Availability

      Catalog NumberAvailability Packaging Qty/Pack Price Quantity
      207008-500UG
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          Glass bottle 500 μg
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          Description
          OverviewAn excellent peptide substrate for calcineurin (Protein Phosphatase 2B, PP2B; Cat. No. 539568). The sequence is derived from the PKA regulatory subunit type II (RII) and is phosphorylated on the Ser residue. Release of phosphate can be quantified colorimetrically using the malachite green method.
          Catalogue Number207008
          Brand Family Calbiochem®
          SynonymsPP2B Substrate, RII Phosphopeptide, DLDVPIPGRFDRRVpSVAAE
          References
          ReferencesDonella-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.
          Fruman, D.A., et al. 1992. Proc. Natl. Acad. Sci. USA 89, 3686.
          Product Information
          ATP CompetitiveN
          FormLyophilized
          FormulationSupplied as a trifluoroacetate salt.
          Hill FormulaC₉₂H₁₅₁N₂₈O₃₂P
          Chemical formulaC₉₂H₁₅₁N₂₈O₃₂P
          Hygroscopic Hygroscopic
          ReversibleN
          Applications
          Biological Information
          Primary TargetCalcineurin (Protein Phosphatase 2B, PP2B)
          Purity≥95% by HPLC
          Physicochemical Information
          Cell permeableN
          Peptide SequenceH-Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser(PO₃H₂)-Val-Ala-Ala-Glu-OH
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          Product Usage Statements
          Storage and Shipping Information
          Ship Code Ambient Temperature Only
          Toxicity Standard Handling
          Storage -20°C
          Hygroscopic Hygroscopic
          Do not freeze Ok to freeze
          Special InstructionsFollowing reconstitution, aliquot and freeze at -20°C. Stock solutions are stable for up to 3 months at -20°C.
          Packaging Information
          Transport Information
          Supplemental Information
          Specifications

          Documentation

          SDS

          Title

          Safety Data Sheet (SDS) 

          Certificates of Analysis

          TitleLot Number
          207008

          References

          Reference overview
          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.
          Fruman, D.A., et al. 1992. Proc. Natl. Acad. Sci. USA 89, 3686.

          Brochure

          Title
          Protein Phosphatases Technical Bulletin
          Data Sheet

          Note that this data sheet 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.

          Revision27-May-2008 RFH
          SynonymsPP2B Substrate, RII Phosphopeptide, DLDVPIPGRFDRRVpSVAAE
          DescriptionAn excellent peptide substrate for calcineurin (Protein Phosphatase 2B, PP2B, Cat. No. 539568). The sequence is derived from the PKA regulatory subunit type II (RII) and is phosphorylated on the Ser residue. Release of phosphate can be quantified colorimetrically using malachite green method.
          FormLyophilized
          FormulationSupplied as a trifluoroacetate salt.
          Recommended reaction conditions
          Malachite Green Assay Note: This protocol is provided only as a general guide. Researchers should standardize this assay for their own specific needs and should consult published literature. Reagents 4.2% Ammonium molybdate: prepare a 4.2% (w/v) solution of ammonium molybdate in 4 M HCl Malachite Green: prepare a 0.045% (w/v) solution of malachite green Malachite Green Solution: Mix 1 volume of 4.2% ammonium molybdate (prepared as above) with 3 volumes of 0.045% malachite green (as prepared above); stir for a minimum of 30 min; filter sterilize using a 0.22 µm filter. This solution is stable at 4°C for 6 months. Malachite Green/Tween Solution: 0.01% Tween®-20 detergent (v/v) is added to an aliquot of Malachite Green Solution just prior to use 2X assay buffer: 200 mM NaCl, 100 mM Tris, 12 mM MgCl2, 1 mM CaCl2, 1 mM DTT, 0.05% NP-40, pH 7.5. Assay buffer can be stored in aliquots at -70°C. Calcineurin Substrate: Prepare a 750 µM (1.64 mg/ml) stock solution in H2O Calmodulin, Bovine Brain (Cat. No. 208690 or 208694): prepare a 25 µM stock solution in H2O. Prepare a working solution of calmodulin by diluting the stock solution 1:50 in 2X assay buffer; a total of 25 µl is needed per reaction. Cell lysate or purified PP2B (Calcineurin) (Cat. No. 539568) Phosphatase inhibitors, as desired 1/2 volume plate KH2PO4: prepare a phosphate standard stock solution by dissolving in 0.05 N HCl to an appropriate concentration of phosphorous (nmol or pmol/ml). Prepare serial dilutions just prior to use for generation of a standard curve. Use 25 µl of each standard dilution plus 25 µl of 2X assay buffer; add 100 µl of Malachite Green/Tween Solution and measure absorbance (see below). Plate reader capable of measuring absorbance at 620 nm Protocol Prepare phosphate standard samples in the desired number of wells in a microtiter plate. It is recommended that the standards be performed in duplicate. Prepare sample wells: 25 µl of 2X assay buffer 10 µl of Calcineurin Substrate 5 µl of Calcineurin preparation (cell lysate or ~40 U of purified enzyme) Note: control samples may be included by the addition of appropriate phosphatase inhibitors. Adjust the H2O volume appropriately to maintain a 50 µl total reaction volume. Incubate for the desired time (e.g. 30 min at 30°C) Terminate the reactions by adding 100 µl of Malachite Green/Tween Solution to each well. Continue incubating for 15-30 min. Read the absorbance at 620 nm. Compare samples to the standard curve to determine phosphatase activity.
          Chemical formulaC₉₂H₁₅₁N₂₈O₃₂P
          Peptide SequenceH-Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser(PO₃H₂)-Val-Ala-Ala-Glu-OH
          Purity≥95% by HPLC
          SolubilityH₂O (2 mg/ml)
          Storage -20°C
          Hygroscopic
          Do Not Freeze Ok to freeze
          Special InstructionsFollowing reconstitution, aliquot and freeze at -20°C. Stock solutions are stable for up to 3 months at -20°C.
          Toxicity Standard Handling
          ReferencesDonella-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.
          Fruman, D.A., et al. 1992. Proc. Natl. Acad. Sci. USA 89, 3686.