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

171536
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      Overview

      Replacement Information

      Pricing & Availability

      Catalogue NumberAvailability Packaging Qty/Pack Price Quantity
      US1171536-5UG
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      In Stock 
      Discontinued
      Limited Quantities Available
      Availability to be confirmed
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          Plastic ampoule 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.
          Applications
          Biological Information
          Purity≥95% 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

          Documentation

          Certificates of Analysis

          TitleLot Number
          171536

          References

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

          Revision11-June-2013 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≥95% by SDS-PAGE
          Storage ≤ -70°C
          Avoid freeze/thaw
          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.