207008 Calcineurin Substrate - Calbiochem

207008
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207008-500UG
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      Plastic ampoule 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
      Quality LevelMQ100
      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

      Calcineurin Substrate - Calbiochem SDS

      Title

      Safety Data Sheet (SDS) 

      Calcineurin Substrate - Calbiochem 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.