HIPK3, His-tag Recombinant

Catalog #

40110

$410 *

Size: 10 µg

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Description

Recombinant human HIPK3 (homeodomain interacting protein kinase 3), encompassing amino acids 163-562. This construct contains an N-terminal His-tag. The recombinant protein was affinity purified and is active.

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Synonyms

PKY, YAK1, DYRK6, FIST3

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Product Data Gallery

4-20% SDS-PAGE Coomassie Staining

Specific Activity

Product Info

Storage and Usage

Citations

Species

Human

Construct

HIPK3 (His-163-562)

Host Species/Expression System

Sf9 insect cells

Purity

≥85%

Format

Aqueous buffer solution

Formulation

50 mM Sodium Phosphate, pH 7.0, 300 mM NaCl, 150 mM Imidazole, 0.1 mM PMSF, 0.25 mM DTT, 25% glycerol

49 kDa

Amino Acids

163–562

Specific Activity

48 pmol/min/µg

Genbank #

NM_005734

UniProt #

Q9H422

Tag(s)

N-terminal His-tag

Background

HIPK3 (homeodomain-interacting protein kinase 3) is a serine/threonine kinase that belongs to the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family, meaning they can phosphorylate themselves and other substrates. Its activity is controlled by several post-translational modifications, such as acetylation, ubiquitination, SUMOylation and caspase cleavage, in addition to phosphorylation. HIPK3 is involved in rRNA transcription and negative regulation of apoptosis and found mainly in the nucleus. HIPKs are involved in chronic fibrosis, Alzheimer’s and Huntington’s disease, and in cancer it was found that its levels have a prognostic value, with its downregulation in non-small cell lung cancer (NSCLC) having poor survival. Advances in RNA deep sequencing have identified circRNA (circular RNAs), which act as miRNA sponges and in the development and progression of cancer. cirHIPK3 (circRNA for HIPK3) is abundant and can sponge miR-124 and induce proliferation of cancer cells. Inhibition of the protein itself or its related circRNA has great potential in cancer oncology.

Storage/Stability

At least 6 months at -80°C.

Assay Conditions

HIPK3 activity was measured by using as substrate the MBP (myelin basic protein) diluted in distilled water to a working concentration of 1 mg/ml, in a [33P]-ATP based assay. Reaction was initiated by mixing increasing amounts of HIPK3 with 1250 pmoles of [33P]-ATP in 5 mM MOPS, pH 7.2, 2.5 mM β-glycerol-phosphate, 5 mM MgCl₂, 1 mM EGTA, 0.4 mM EDTA, 50 ng/µl BSA, 50 µM DTT and substrate at a final concentration of 200 µg/ml.

The reaction was initiated by addition of [33P]-ATP Assay Cocktail (50 µM of [33P]-ATP with 50 µM ATP), 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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Datasheets/Protocols

40110 Lot 231106

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