MAPKAPK5, GST-tag Recombinant

Catalog #

40118

$410 *

Size: 10 µg

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Description

Recombinant human full length MAPKAPK5 (MAP kinase-activated protein kinase 5). This construct contains an N-terminal GST-tag. The recombinant protein was affinity purified and is active.

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Synonyms

PRAK

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Product Data Gallery

4-20% SDS-PAGE Coomassie Staining

Specific Activity

Product Info

Storage and Usage

Citations

Species

Human

Construct

MAPKAPK5 (GST-Full Length)

Host Species/Expression System

Sf9 insect cells

Purity

≥80%

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

79 kDa

Amino Acids

full length

Specific Activity

172 pmol/min/µg

Genbank #

NM_003668

UniProt #

Q8IW41

Tag(s)

N-terminal GST-tag

Background

MAPKAPK5 is a member of the serine/threonine kinase family that responds to cellular stress and proinflammatory cytokines. MAPKAPK5 is activated through its phosphorylation by MAP kinases including MAPK1/ERK, MAPK14/p38-alpha, and MAPK11/p38-beta (1). MAPKAPK5 is activated in HeLa cells in response to cellular stress and proinflammatory cytokines. MAPKAPK5 activity is regulated by p38-alpha and p38-beta both in vitro and in vivo, and thr-182 is the regulatory phosphorylation site of MAPKAPK5 (2). In vitro, MAPKAPK5 kinase phosphorylates heat shock protein HSP27 at its physiologically relevant sites.

References

1. New, L.; Jiang, Y. et al: PRAK, a novel protein kinase regulated by the p38 MAP kinase. EMBO J. 17: 3372-3384, 1998.
2. Ni, H.; Wang. et al: MAPKAPK5, a novel mitogen-activated protein kinase (MAPK)-activated protein kinase, is a substrate of the extracellular-regulated kinase (ERK) and p38 kinase. Biochem. Biophys. Res. Commun. 243: 492-496, 1998.

Storage/Stability

At least 6 months at -80°C.

Assay Conditions

MAPKAPK5 activity was measured by using the HSP27tide synthetic peptide (RRLNRQLSVA-amide) at a working concentration of 1 mg/ml, in a [33P]-ATP based assay. Reaction was initiated by mixing increasing amounts of MAPKAPK5 with 1250 pmoles of [33P]-ATP in 5 mM MOPS, pH 7.2, 2.5 mM β-glycerol-phosphate, 5 mM MgCl₂, 2.5 mM MnCl₂, 1 mM EGTA, 0.4 mM EDTA, 50 ng/µl BSA prepared with 50 µM DTT, 50 μM ATP and substrate at a final concentration of 200 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, air-dry and 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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40118 Lot 230629

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