IR Human RTK Kinase Enzymatic Radiometric LeadHunter Assay [10 μM ATP] - TW

Item: 174990

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OVERVIEW

DOCUMENTS/REFERENCES

Biological Information

Background Information:

The insulin receptor kinase (IRK) is composed of two extracellular a-subunits (135 kDa), which contain the insulin-binding site, and two intracellular b-subunits (95 kDa), containing the protein tyrosine kinase domain. After insulin binding, the insulin-IRK complex internalizes into muscle and fat cells. The IRK substrates of IRS-1 and IRS-2 form complexes, through Src homology region 2 (SH2) domains, with docking molecules such as phosphoinositde-3-kinase (PI3K). The recruitment of PI3K in turn activates phosphoinositide-dependent kinases, which serve in the activation of protein kinase B (Akt) and atypical isoforms of protein kinase C (PKC). These activated kinases then phosphorylate downstream effectors, ultimately promoting the translocation of insulin-sensitive glucose transporter subtype 4 (GLUT-4) to the plasma membrane, to operate in the coordination of glucose metabolism.

Family:

Kinase

Sub Family:

RTK

Class:

Protein Tyrosine

Protein Name:

Uniprot Number:

P06213

Protein Aliases:

CD antigen CD220 [Cleaved into: Insulin receptor subunit alpha; Insulin receptor subunit beta]

Gene Name:

INSR

Gene ID:

3643

Gene Aliases:

CD220

Accession Number:

NM_000208

Organism:

Human

Construct Details:

Fragment (1011-1382), Recombinant

Assay Information

Assay Type:

Biochemical

Assay Sub Type:

Enzymatic

Functional Mode:

Antagonist

Detection Method:

Radiometry

Measured Response:

Scintillation

Testing Information

Procedure Summary:

Human recombinant protein kinase Insulin Receptor expressed in insect cells is used. Test compound and/or vehicle is preincubated with 0.067 µg/ml enzyme‡ for 15 minutes at 37°C in modified HEPES buffer pH 7.4. The reaction is initiated by addition of 0.2 mg/ml poly(Glu:Tyr), 10 μM ATP containing 0.25 μCi [γ32P]ATP for 30 minutes and terminated by further addition of 3% H3PO4. An aliquot is counted to determine the radioactivity of [32P]Poly(Glu:Tyr). Compounds are screened at 10 μM.

Substrate:

200 µg/ml Poly(Glu:Tyr)

ATP Concentration:

10 uM

Incubation:

30 min at 37°C

Control Inhibitor:

GSK 1838705

Criteria For Significance:

≥ 50% of max stimulation or inhibition

Test Concentration / Dose:

Suggested concentration for initial testing is 10 uM

Test Sample Requirements:

Minimum for 1) Screen: 25 μl of 10 mM stock -OR- 1 mg (pre-weighed). 2) Dose Response: 60 μl of 10 mM stock -OR- 1 mg (pre-weighed).

Minimum Order Quantity:

Turnaround Time

Standard:

15 Business Days

Reference Compound Data

PubChem ID

122130844

Name

Staurosporine

Measurement

0.24μM -

PubChem ID

25182616

Name

GSK 1838705

Measurement

0.0068μM -

Clinical Relevance

Therapeutic Area:

Metabolic Diseases

Adverse / Beneficial Action:

The insulin receptor kinase may be effective in improving glycemic control in diabetics.

Additional Information

Brand:

LeadHunter

Testing Location:

Taiwan - Taipei