EEF2K, GST-tag Recombinant

Catalog #

40057

$410 *

Size: 10 µg

Qty

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Description

Recombinant full-length human EEF2K was expressed in Sf9 insect sells using an N-terminal GST-tag.

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Synonyms

eEF-2K, HSU93850, MGC45041

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Product Data Gallery

4-20% SDS-PAGE Coomassie Staining

Specific Activity

Product Info

Storage and Usage

Citations

Species

Human

Construct

EEF2K (GST-Full Length)

Host Species/Expression System

Sf9 insect cells

Purity

≥70%

Format

Aqueous buffer solution

Formulation

50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM Glutathione, 0.1 mM EDTA, 0.25 mM DTT, 0.1 mM PMSF, 25% glycerol

125 kDa

Amino Acids

full length

Genbank #

NM_013302

UniProt #

O00418

Tag(s)

N-terminal GST-tag

Background

EEF2K is a highly conserved calmodulin-dependent protein kinase that links activation of cell surface receptors to cell division. EEF2K is involved in the regulation of protein synthesis. It phosphorylates eukaryotic elongation factor 2 (EEF2), an abundant cytoplasmic protein that catalyzes the movement of the ribosome along mRNA during translation in eukaryotic cells, and inhibits the EEF2 function (1). EEF2K is highly expressed in heart and skeletal muscle, suggesting that EEF2 phosphorylation may be particularly important in muscle. EEF2K is highly expressed in patients with systemic lupus erythematosus (2) as well as in many cancers.

References

1. Ryazanov, A. G. et al; Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase. Proc. Nat. Acad. Sci. 94: 4884-4889, 1997.
2. Arora, S. et al; Detection of anti-elongation factor 2 kinase (calmodulin-dependent protein kinase III) antibodies in patients with systemic lupus erythematosus. Biochem. Biophys. Res. Commun. 293: 1073-1076, 2002.

Storage/Stability

At least 6 months at -80°C.

Assay Conditions

EEF2K activity was measured by using the Ef2tide synthetic peptide substrate (RKKFGESEKTKTKEFL), diluted in distilled water to a final concentration of 1 mg/ml, in a [33P]-ATP based assay. Reaction was initiated by mixing increasing amounts of the EEF2K with 1250 pmoles of [33P]-ATP in 25 mM MOPS, pH 7.2, 12.5 mM β-glycerol-phosphate. 25 mM MgCl₂, 5 mM EGTA, 2 mM EDTA prepared with 250 µM DTT, co-factor calcium/calmodulin and substrate at a final concentration of 250 µg/ml.

The reaction was initiated by addition of [33P]-ATP Assay Cocktail, followed by a 15-minute incubation at 30°C. The reaction was terminated by spotting the reaction mixture on phosphocellulose P81 paper, followed by three 10-minute washes with 1% phosphoric acid solution. Radioactivity was measured in a scintillation counter. The corrected activity (RLU) was calculated by removing the blank value for each sample. The Kinase Specific Activity was calculated as follows: RLU / [(specific activity of [33P]-ATP in cpm/pmol)*(Reaction time in min)*(Enzyme amount in µg or mg)] * [(Reaction Volume) / (Spot Volume)]. The blank was determined from a “no substrate” sample by replacing the substrate solution with an equal volume of distilled water.

Instructions for Use

Thaw on ice and gently mix prior to use. DO NOT VORTEX. Perform a quick spin before opening. Aliquot into small volumes and flash freeze for long term storage. Avoid multiple freeze/thaw cycles.

Applications

Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.

Shipping Temperature

-80°C

Warnings

Avoid freeze/thaw cycles

Scientific Category

Kinase/Serine-Threonine

Regulatory Status

Research Use Only

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Datasheets/Protocols

40057 Lot 230502

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