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PK02 Raytide™ Substrate

Raytide™ Substrate MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
PK02
  
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      Overview

      Replacement Information

      Key Spec Table

      Purity
      >98% by SDS-PAGE
      Description
      OverviewModified gastrin analog that serves as a general substrate for tyrosine kinases. Contains one phosphorylation site per molecule.
      Catalogue NumberPK02
      Brand Family Calbiochem®
      References
      ReferencesGuan, K.L., et al. 1991. Nature 350, 359.
      Product Information
      FormLyophilized
      FormulationLyophilized in a volatile buffer, BSA.
      Positive controlProtein-Tyrosine Kinase, (p60c-src), Cat. No. PK03, and Raytide™, Cat. No. PK02, a substrate for tyrosine kinases, can serve as positive controls in experiments measuring protein phosphorylation.
      Applications
      Application NotesKinase Assay
      Application CommentsActivity of p60c-src can be measured as described below using Raytide™, other peptide substrates and many naturally occurring proteins. This preparation of p60c-src has no detectable Ser or Thr kinase activity. Suggested Kinase Reaction Protocol Materials required: [γ-32P]ATP (10 mCi/ml). Kinase Assay Buffer: 50 mM HEPES, pH 7.5 containing 0.1 mM EDTA and 0.015% Brij 35. Kinase Dilution Buffer: kinase assay buffer containing 0.1 mg/ml BSA and 0.2% β-mercaptoethanol (note: β-mercaptoethanol is required for src kinase activity). ATP Mix: 0.15 mM ATP, 30 mM MgCl2 and 200 mCi/ml [γ-32P]ATP in kinase assay buffer. Phosphoric acid Acetone P81 Ion Exchange Chromatography paper (i.e. Whatman No. 3698915). Procedure: 1. Dilute p60c-src stock solution to 0.2 units/μl in Kinase Dilution Buffer. 2. Mix 10 μl diluted p60c-src with 10 μl Raytide™ (1 mg/ml solution in Kinase Assay Buffer). 3. Start the reaction by adding 10 μl of ATP Mix. 4. Incubate at 30°C for 30 minutes. 5. Stop the reaction by adding 120 μl of 10% phosphoric acid. Vortex and apply 120 μl onto a square (2 cm x 2 cm) of P81 paper. 6. Wash papers extensively in 0.5% phosphoric acid. Wash once with acetone. Dry and quantitate by liquid scintillation counting. (Phosphorylated Raytide™ will stick, while ATP will wash off the papers). Notes: Each unit of p60c-src enzyme catalyzes the incorporation of one pmol phosphate per minute from ATP into tyrosyl residues. In addition to phosphorylating Raytide™, the p60c-src protein-tyrosine kinase can be used to phosphorylate many naturally occurring proteins. Natural substrates phosphorylated on tyrosyl residues may be purified from complex mixtures using the Immunoaffinity System for purification of phosphotyrosyl proteins (Cat. No. PTS01). In addition to being phosphorylated by p60c-src, Raytide™ can be used as a substrate for many naturally occurring tyrosine kinases. Raytide™ contains BSA. Raytide™ control peptide (without a tyrosine, Cat. No. PK05) is also available. To use Raytide™ in a dephosphorylation assay with tyrosine phosphatases, see Nature 350, 359-62, 1991.
      Biological Information
      Purity>98% by SDS-PAGE
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Standard Handling
      Storage +2°C to +8°C
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Special InstructionsFollowing reconstitution, aliquot and freeze (-20°C) or refrigerate (4°C) with 0.1% azide. Stock solutions are stable for up to 3 months at -20°C. Avoid freeze/thaw cycles of solutions.
      Packaging Information
      Transport Information
      Supplemental Information
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      PK02 0

      Documentation

      Raytide™ Substrate Certificates of Analysis

      TitleLot Number
      PK02

      References

      Reference overview
      Guan, K.L., et al. 1991. Nature 350, 359.

      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.

      Revision18-September-2008 RFH
      ApplicationKinase Assay
      DescriptionRaytide™ is a modified gastrin analog. There is one tyrosyl phosphorylation site per Raytide™ molecule. The carboxyl end of the peptide contains acidic and hydrophilic amino acid residues, while the amino terminus contains basic amino acids. In phosphoric acid the amino end is positively charged and binds to phosphocellulose paper (p81 Whatman). The distance between the two charged ends of the peptide prevents neutralization. Additionally, the peptide maintains an unfolded state and functions as an excellent substrate for protein-tyrosine kinases and tyrosine phosphatases (Nature 350, 359-62, 1991). Raytide™ EL, Cat. No. PK04, which has a higher labeling efficiency when used as either a kinase or a phosphatase substrate is also available. Note: the exact sequence of Raytide™ and Raytide™ EL is proprietary to Calbiochem and cannot be released.
      BackgroundRaytide™ is a modified gastrin analog. There is one tyrosyl phosphorylation site per Raytide™ molecule. The carboxyl end of the peptide contains acidic and hydrophilic amino acid residues, while the amino terminus contains basic amino acids. In phosphoric acid the amino end is positively charged and binds to phosphocellulose paper (p81 Whatman). The distance between the two charged ends of the peptide prevents neutralization. Additionally, the peptide maintains an unfolded state and functions as an excellent substrate for protein-tyrosine kinases and tyrosine phosphatases (Nature 350, 359-62, 1991). Raytide™ EL, Cat. No. PK04, which has a higher labeling efficiency when used as either a kinase or a phosphatase substrate is also available. Note: the exact sequence of Raytide™ and Raytide™ EL is proprietary to Calbiochem and cannot be released.
      Positive controlProtein-Tyrosine Kinase, (p60c-src), Cat. No. PK03, and Raytide™, Cat. No. PK02, a substrate for tyrosine kinases, can serve as positive controls in experiments measuring protein phosphorylation.
      FormLyophilized
      FormulationLyophilized in a volatile buffer, BSA.
      Recommended reaction conditionsSuggested Kinase Protocol Materials required: • [γ-32P]ATP (10 mCi/ml). • Kinase Assay Buffer: 50 mM HEPES, pH 7.5 containing 0.1 mM EDTA and 0.015% Brij®-35 detergent. • Kinase Dilution Buffer: kinase assay buffer containing 0.1 mg/ml BSA and 0.2% β-mercaptoethanol (note: β-mercaptoethanol is required for src kinase activity). • ATP Mix: 0.15 mM ATP, 30 mM MgCl2 and 200 mCi/ml [γ-32P]ATP in kinase assay buffer. • Phosphoric acid • Acetone • P81 Ion Exchange Chromatography paper (i.e. Whatman No. 3698915). Procedure: 1. Dilute p60c-src stock solution to 0.2 units/µl in Kinase Dilution Buffer. 2. Mix 10 µl diluted p60c-src with 10 µl Raytide™ (1 mg/ml solution in Kinase Assay Buffer). 3. Start the reaction by adding 10 µl of ATP Mix. 4. Incubate at 30°C for 30 min. 5. Stop the reaction by adding 120 µl of 10% phosphoric acid. Vortex and apply 120 µl onto a square (2 cm x 2 cm) of P81 paper. 6. Wash papers extensively in 0.5% phosphoric acid. Wash once with acetone. Dry and quantitate by liquid scintillation counting. (Phosphorylated Raytide™ will stick, while ATP will wash off the papers). Notes: In addition to being phosphorylated by p60c-src, Raytide™ can be used as a substrate for many naturally occurring tyrosine kinases. Raytide™ control peptide (without a tyrosine, Cat. No. PK05) is also available. To use Raytide™ in a dephosphorylation assay with tyrosine phosphatases, see Nature 350, 359-62, 1991.
      Purity>98% by SDS-PAGE
      SolubilityAqueous Buffers (1 mg/ml)
      CommentsActivity of p60c-src can be measured as described below using Raytide™, other peptide substrates and many naturally occurring proteins. This preparation of p60c-src has no detectable Ser or Thr kinase activity. Suggested Kinase Reaction Protocol Materials required: [γ-32P]ATP (10 mCi/ml). Kinase Assay Buffer: 50 mM HEPES, pH 7.5 containing 0.1 mM EDTA and 0.015% Brij 35. Kinase Dilution Buffer: kinase assay buffer containing 0.1 mg/ml BSA and 0.2% β-mercaptoethanol (note: β-mercaptoethanol is required for src kinase activity). ATP Mix: 0.15 mM ATP, 30 mM MgCl2 and 200 mCi/ml [γ-32P]ATP in kinase assay buffer. Phosphoric acid Acetone P81 Ion Exchange Chromatography paper (i.e. Whatman No. 3698915). Procedure: 1. Dilute p60c-src stock solution to 0.2 units/μl in Kinase Dilution Buffer. 2. Mix 10 μl diluted p60c-src with 10 μl Raytide™ (1 mg/ml solution in Kinase Assay Buffer). 3. Start the reaction by adding 10 μl of ATP Mix. 4. Incubate at 30°C for 30 minutes. 5. Stop the reaction by adding 120 μl of 10% phosphoric acid. Vortex and apply 120 μl onto a square (2 cm x 2 cm) of P81 paper. 6. Wash papers extensively in 0.5% phosphoric acid. Wash once with acetone. Dry and quantitate by liquid scintillation counting. (Phosphorylated Raytide™ will stick, while ATP will wash off the papers). Notes: Each unit of p60c-src enzyme catalyzes the incorporation of one pmol phosphate per minute from ATP into tyrosyl residues. In addition to phosphorylating Raytide™, the p60c-src protein-tyrosine kinase can be used to phosphorylate many naturally occurring proteins. Natural substrates phosphorylated on tyrosyl residues may be purified from complex mixtures using the Immunoaffinity System for purification of phosphotyrosyl proteins (Cat. No. PTS01). In addition to being phosphorylated by p60c-src, Raytide™ can be used as a substrate for many naturally occurring tyrosine kinases. Raytide™ contains BSA. Raytide™ control peptide (without a tyrosine, Cat. No. PK05) is also available. To use Raytide™ in a dephosphorylation assay with tyrosine phosphatases, see Nature 350, 359-62, 1991.
      Storage Avoid freeze/thaw
      +2°C to +8°C
      Do Not Freeze Ok to freeze
      Special InstructionsFollowing reconstitution, aliquot and freeze (-20°C) or refrigerate (4°C) with 0.1% azide. Stock solutions are stable for up to 3 months at -20°C. Avoid freeze/thaw cycles of solutions.
      Toxicity Standard Handling
      ReferencesGuan, K.L., et al. 1991. Nature 350, 359.

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