Recombinant human WNKS3 (WNK lysine deficient protein kinase 3), encompassing amino acids (1-571). This construct contains an N-terminal GST-tag. The recombinant protein was affinity purified.
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Synonyms
Protein Kinase With No Lysine 3, WNK Lysine Deficient Protein Kinase 3, Protein Kinase, Lysine Deficient 3
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
MW
91 kDa
Amino Acids
1-571
Specific Activity
≥13 pmol/min/μg
Genbank #
NM_001002838
UniProt #
Q9BYP7
Tag(s)
N-terminal GST-tag
Background
WNK3 is a member of serine-threonine protein kinases, which was named the with-no-lysine family. WNK3 is expressed in several tissues including kidney and brain. WNK3 can regulate the activity of the electroneutral cation-coupled chloride cotransporters by activating NKCC1/2 and NCC and inhibiting KCCs. WNK3 kinase function is required for normal blood pressure homeostasis and the control of neuronal excitability. Also, overexpression of WNK3 increases the survival of Hela cells through caspase-3 pathway.
Storage/Stability
At least 6 months at -80°C.
Assay Conditions
The kinase activity of the complex was measured using the ADP-Glo™ Kinase Assay kit (Promega; Cat#V9101) which quantifies the amount of ADP produced. The ADP-Glo™ Reagent is added to terminate the reaction and deplete the remaining ATP. The Kinase Detection Reagent is then added to convert ADP to ATP and to measure the newly synthesized ATP using a luciferase reaction.
Kinase activity was measured by Myelin basic protein (MBP) diluted in water to a final concentration of 0.5 mg/ml. Reaction was initiated by mixing increasing amounts of the WNK3 with 0.125 mM ATP in 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 0.1 mg/ml BSA, 250 µM DTT with the 0.5 mg/ml substrate and 12.5 mM MnCl2.
After a 40-minute incubation at 37°C, the reaction was terminated by addition of the AMP-Glo™ Reagent followed by a subsequent 40 minute incubation at room temperature. Kinase Detection Reagent was then added and incubated for another 30 minutes. Detection of luminescence was measured using the Luminescence Module Protocol on GloMax®-Multi Micorplate reader. The corrected activity (RLU) was calculated by removing the blank value for each sample divided by the (specific activity of ADP in RLU/pmol)*(Reaction time in min)*(Enzyme amount in µg or mg). The blank was determined from a “no kinase” sample by replacing the enzyme working solution with an equal volume of Kinase Dilution Buffer IX (1X).
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
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