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171536 AMPK (α1/β1/γ1), His•Tag®, Human, Recombinant, S. frugiperda

AMPK (α1/β1/γ1), His•Tag®, Human, Recombinant, is a full length enzyme. Consists of subunits A1, B1, & G1, each fused at the C-terminus to a His•Tag® sequence & co-expressed in S. frugiperda.

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171536
Preis & Verfügbarkeit

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Preis & Verfügbarkeit

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171536-5UG
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      		 Kst.-Ampulle 5 μg
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      Description
      OverviewFull length, recombinant, human AMPK subunits (A1, B1, and G1), each fused at the C-terminus to a His•Tag® sequence and co-expressed in S. frugiperda insect cells using a baculovirus expression system. AMPK is involved in sensing energy levels in the cell that result from changes in AMP levels. AMPK is an upstream regulator of mTOR activation. The active complex is useful in the study of kinase activity regulation and inhibitor screening.
      M.W. = 68,000 (A1), 38,000 (B1), 40,000 (G1)
      Catalogue Number171536
      Brand Family Calbiochem®
      Application Data

      The specific activity of AMPK was measured as outlined in the Recommended Reaction Conditions.
      References
      ReferencesScott J.W., 2007. EMBO J 26, 806.
      Minokoshi Y et al. 2004. Nature 428, 569.
      Product Information
      FormLiquid
      FormulationIn 300 mM NaCl, 150 mM Imidazole, 50 mM Sodium Phosphate, 0.2 mM DTT, 0.1 mM PMSF, 25% glycerol, pH 7.0.
      Quality LevelMQ100
      Applications
      Biological Information
      Purity≥90% 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 Dry Ice Only
      Toxicity Standard Handling
      Storage ≤ -70°C
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
      Packaging Information
      Transport Information
      Supplemental Information
      Specifications
      Global Trade Item Number
      Bestellnummer GTIN
      171536-5UG 04055977207248

      Documentation

      AMPK (α1/β1/γ1), His•Tag®, Human, Recombinant, S. frugiperda SDB

      Titel

      Sicherheitsdatenblatt (SDB) 

      AMPK (α1/β1/γ1), His•Tag®, Human, Recombinant, S. frugiperda Analysenzertifikate

      TitelChargennummer
      171536

      Literatur

      Übersicht
      Scott J.W., 2007. EMBO J 26, 806.
      Minokoshi Y et al. 2004. Nature 428, 569.
      Datenblatt

      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.

      Revision03-August-2020 JSW
      Application Data

      The specific activity of AMPK was measured as outlined in the Recommended Reaction Conditions.
      DescriptionFull length, recombinant, human AMPK subunits (A1, B1, and G1), each fused at the C-terminus to a His•Tag® sequence and co-expressed in S. frugiperda insect cells using a baculovirus expression system. AMPK is involved in sensing energy levels in the cell that result from changes in AMP levels. AMPK is an upstream regulator of mTOR activation. The active complex is useful in the study of kinase activity regulation and inhibitor screening. M.W. = 68,000 (A1), 38,000 (B1), 40,000 (G1).
      FormLiquid
      FormulationIn 300 mM NaCl, 150 mM Imidazole, 50 mM Sodium Phosphate, 0.2 mM DTT, 0.1 mM PMSF, 25% glycerol, pH 7.0.
      Recommended reaction conditions
      Activity Assay Materials Required • Kinase Assay Buffer: 25 mM MOPS, 25 mM MgCl2, 12.5 mM β-glycerophosphate, 5 mM EGTA, 2 mM EDTA, pH 7.2; add 0.25 mM DTT just prior to use • Kinase Dilution Buffer: Kinase Assay Buffer diluted 1:5 with 50 ng/µl BSA, 5% glycerol solution • AMPK: Prepare serial dilutions using Kinase Dilution Buffer as outlined in the sample data below or as deemed necessary for individual experimental conditions • 10 mM ATP Stock Solution: Dissolve 55 mg ATP in 10 ml Kinase Assay Buffer; dispense into 200 µl aliquots and freeze (-20°C) • 250 µM [32P]-ATP Assay Cocktail: 150 µl 10 mM ATP stock solution, 100 µl [32P]-ATP (1 mCi/100 µl), 5.75 ml Kinase Assay Buffer; dispense into 1 ml aliquots and freeze (-20°C) • Substrate: Dissolve SAMS substrate (HMRSAMSGLHLVKRR) in dH2O to a final concentration of 1 mg/ml • 0.5 mM AMP Solution: Prepare a 0.5 mM solution of AMP
      Activity Assay Protocol 1. Thaw an aliquot of [32P]-ATP Assay Cocktail in a shielded container in a designated radioactive working area. 2. Thaw the AMPK, Kinase Assay Buffer, Substrate, and Enzyme Dilution Buffer on ice. 3. In a pre-cooled tube, add the following components: • 10 µl diluted AMPK • 5 µl 1 mg/ml Substrate stock • 5 µl 0.5 mM AMP solution 4. Prepare a Blank as outlined in step 3, replacing the 5 µl Substrate with an equal volume of water. 5. Initiate the reaction by adding 5 µl [32P]-ATP Assay Cocktail (final volume is 25 µl) and incubating in a water bath at 30°C for 15 min. 6. Terminate the reaction by spotting 20 µl of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper. 7. Air-dry the pre-cut strips and sequenentially wash in 1% phosphoric acid with constant, gentle stirring for 10 min. Repeat for a total of 3 washes. 8. Count the radioactivity in presence of scintillation fluid. 9. Determine the corrected cpm by substracting the value of the Blank from each sample and calculate as follows:

      Figure 1: Calculation of Activity
      Purity≥90% by SDS-PAGE
      Storage Avoid freeze/thaw
      ≤ -70°C
      Do Not Freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
      Toxicity Standard Handling
      ReferencesScott J.W., 2007. EMBO J 26, 806.
      Minokoshi Y et al. 2004. Nature 428, 569.