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Phosphoinositide 3-kinase p110γ (human), (recombinant)

 
ALX-201-055-C010 10 µg 410.00 USD
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Product Specification

Alternative Name:PI(3)K p110γ
 
Source:Produced in Sf9 cells. Full length PI(3)K p110γ (human phosphoinositide-3-kinase p110γ) is fused at the N-terminus to a His-tag.
 
EC:2.7.1.137
 
UniProt ID:P48736
 
Concentration:1mg/ml  
 
Formulation:Liquid. In 10mM HEPES, pH 7.5, containing 100mM sodium chloride, 0.5mM magnesium chloride and 50% glycerol.
 
Purity:≥95% (SDS-PAGE)
 
Specific Activity:~4.5nmol/mg/min using phosphatidylinositol as the substrate (lipid kinase activity test).
 
Shipping:Shipped on Dry Ice
 
Long Term Storage:-20°C
 
Protocol:Lipid kinase activity test:
Kinase reaction buffer: 20mM TRIS-HCl, pH 7.4; 4mM MgCl2; 100mM sodium chloride.
- Reaction mixture in total volume of 100ml was:
1x reaction buffer (final concentration), enzyme (4 reactions with 1, 2, 3 or 4µg were performed), 10µg phosphatidylinositol, 20µM cold ATP and 10µCi 32P-ATP.
- Reaction was started by addition of the 32P-ATP and incubate for 10 minutes at 37°C.
- Reaction was stopped by adding 150µl 1M HCl.
- Lipids were extracted with 400µl chloroform:methanol (1:1) by vortexing.
- Extracts were washed three times with 200µl 1M HCl.
- Incorporation of 32P in the extracted lipids was measured by mixing extract with 3ml scintillation cocktail (ROTH) and counting in a liquid scintillation analyzer.
- To determine specific activity of PI3K p110γ the assay was performed under the same conditions stated above by using 10µCi 32P-ATP with a value of 10'000 units.
- Average value of measurements of the lipid extracts of the performed 4 reactions was 227 units for 1µg. Dividing 10’000 by 227 a factor of 44 times less for the incorporation was determined.
-Total ATP (20µM) was divided by 44 giving 0.454µM for 10 minutes reaction time or 45.4M/min/µg. Recalculation of this value for a 1 liter solution results in 4.5 nmoles/min/mg.
 

Product Literature References

Gbetagamma stimulates phosphoinositide 3-kinase-gamma by direct interaction with two domains of the catalytic p110 subuni: D. Leopoldt, et al.; J. Biol. Chem. 273, 7024 (1998), Abstract; Full Text
Linkage of G protein-coupled receptors to the MAPK signaling pathway through PI 3-kinase gamma: M. Lopez-Ilasaca, et al.; Science 275, 394 (1997), Abstract;
Lipid kinase and protein kinase activities of G-protein-coupled phosphoinositide 3-kinase gamma: structure-activity analysis and interactions with wortmannin: S. Stoyanova, et al.; Biochem. J. 324, 489 (1997), Abstract; Full Text
Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase: B. Stoyanov, et al.; Science 269, 690 (1995), Abstract;

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