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

171536
Purchase on Sigma-Aldrich

Descripción

Replacement Information

Products

Número de referenciaEmbalaje Cant./Env.
171536-5UG Ampolla de plást. 5 μg
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

AMPK (α1/β1/γ1), His•Tag®, Human, Recombinant, S. frugiperda Ficha datos de seguridad (MSDS)

Título

Ficha técnica de seguridad del material (MSDS) 

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

CargoNúmero de lote
171536

Referencias bibliográficas

Visión general referencias
Scott J.W., 2007. EMBO J 26, 806.
Minokoshi Y et al. 2004. Nature 428, 569.
Ficha técnica

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.