Promega

PI3K (p110 delta/p85 alpha),

Varenummer: V1771
Kort informasjon 10000
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Phosphatidylinositol (PI) and its phosphorylated derivates, collectively called phosphoinositides, are important second messengers that are critical as signaling molecules and for cellular membrane remodeling. These derivatives are generated by a family of kinases called phosphoinositide lipid kinases (PIKs). Nineteen PIK isoforms have been identified in mammals. Based on their ability to preferentially phosphorylate the hydroxyl group of the inositol ring on position 3, 4 or 5, they have been broadly classified into three major families: phosphoinositide 3-kinases (PI3Ks), phosphoinositide 4-kinases (PI4Ks) and phosphoinositide phosphate-kinases (PIP5Ks and PIP4Ks). Promega lipid kinase enzymes, substrates and detection systems provide a complete set of reagents for performing phosphoinositide lipid kinase (PIK) reactions using a luminescent ADP-detection platform, the ADP-Glo Kinase Assay. The reagents include purified human recombinant proteins of Class I PI3Ks, optimized reaction buffer and ready-to-use lipid kinase substrates. The enzymes are available separately or can be purchased as part of the PI3K-Glo Class I Profiling Kit, which contains PI3Ks (alpha, beta, gamma and delta; 5ug each), PIP2:3PS Lipid Kinase Substrate (0.25mg) and the ADP-Glo Kinase Assay, 1 000 assays. The lipid substrates are supplied as frozen small unilamellar vesicles containing a mixture of phosphatidylinositol (PI) or phosphoinositol-4,5-bisphosphate (PIP2) at a 1:3 ratio with phosphatidylserine (PS) as carrier lipid. A substrate composed of PIP2 and PS at a 1:3 ratio was optimized to use with class I PI3Ks. A substrate composed of PI and PS at a 1:3 ratio was demonstrated to be recognized by the majority of family members and provides a universal PI lipid kinase substrate. The principle of the lipid kinase assay is illustrated in Figure 1. The lipid kinase reaction is performed by incubating lipid substrate (PI:3PS or PIP2:3PS) with a recombinant enzyme and ATP, and the kinase activity is measured using the ADP-Glo Kinase Assay. The ADP-Glo Kinase Assay is performed in two steps. After the kinase reaction, an ATP-depletion reagent is added to terminate the lipid kinase reaction and deplete any remaining ATP, leaving only ADP. Next, a detection reagent is added to simultaneously convert ADP to ATP and allow the newly synthesized ATP to be converted to light using a coupled luciferase/luciferin reaction.|

Employ Complete Solutions for Class I PI3Ks:
Purified human recombinant enzymes with high specific activity.
Ready-to-use lipid substrate (PI or PIP2).
Universal reaction buffer formulation.
Highly sensitive detection assay.
Observe Excellent Selectivity: High signal-to-background ratios even at low % conversion of substrate.
Obtain Reliable Results: The broad dynamic range, low background and excellent sensitivity result in less ambiguous data.
Save Time: Homogeneous assay with simple "add-and-read" format.
Avoid False Hits: The special formulation and luminescent signal results in low false-hit rate.
Save Money: Easily scalable to 1,536-well format, reducing cost per well.

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Ekstra spesifikasjoner
Recombinant PI3K Enzymes: Store recombinant PI3K enzymes below -65 C. At first use, rapidly thaw and place on ice. Dispense any unused material into single-use aliquots and immediately snap-freeze the vials. Avoid multiple freeze-thaw cycles.Lipid Substrates: Store lipid substrates below -65 C. Before use, thaw at room temperature and allow substrate to equilibrate completely to room temperature. Mix extensively by vortexing for at least 1 minute. Thawed lipid substrates can be kept at room temperature (15-30 C) for at least 6 hours or stored at 2-10 C for one week.Buffers: Store 5X PI3K Reaction Buffer, 10X Lipid Dilution Buffer and 1M MgCl2 at -30 C to -10 C.ADP-Glo(TM); Kinase Assay: Upon receiving ADP-Glo(TM); Kinase Assay, remove ATP and store it below -65 C. Store the rest of the components at -30 to -10 C. Before use, thaw all components completely at room temperature. Once thawed, mix each component thoroughly before use. Because ATP is naturally prone to hydrolysis after freeze-thaw cycles, dispense into single-use aliquots and store below -65 C. Once thawed and prepared, dispense Kinase Detection Reagent (Kinase Detection Buffer + Substrate) and ADP-Glo(TM); Reagent into aliquots and store at -30 to -10 C. For convenience, both reagents may be used at room temperature for 24 hours without loss of signal.|For Cat.# V1781, V1782, V1791, V1792:Patents Pending. U.S. Pat. No. 7,700,310 and other patents and patents pending. U.S. Pat. Nos. 6,602,677, 7,241,584 and 8,030,017 and other patents and patents pending.U.S. Pat. Nos. 7,083,911, 7,452,663 and 7,732,128 and other patents. U.S. Pat. No. 7,741,067 and other patents and patents pending. The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and 5,700,673. Licensed from Lonza Nottingham Ltd. under U.S. Pat. Nos. 6,599,711 and 6,911,319 and other pending and issued patents.

Kontaktperson(er) til dette produktet

Christine Rindal Ibra 944 34 009 christine.rindal.ibra@nmas.no
Claudia Emmanuel 951 51 950 claudia.emmanuel@nmas.no
Monica Laukas 404 40 960 monica.laukas@nmas.no

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