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Lipid Kinase Assays: PI3K-Glo™ Class I Profiling Kit

Simple, Add-and-Read Assay to Detect Class I PI3K Activity

  • Includes purified recombinant proteins of class I PI3Ks, optimized reaction buffer, ready-to-use lipid kinase substrates and ADP-Glo™ Kinase Assay
  • High signal-to-background ratios even at low percent substrate conversion
  • Easily scalable to 1,536-well format

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Catalog number selected: V1690

$ 1,240.00
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Lipid Kinase Assays: PI3K-Glo™ Class I Profiling Kit
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$ 1,240.00
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Scalable Assays to Detect Phosphoinositide Kinase Activity

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.

PI3K-Glo™ Class I Profiling Kit

The PI3K-Glo™ Class I Profiling Kit contains PI3Ks (α, β, γ and δ; 5μg 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. The PIP2:3PS substrate is optimized for use with class I PI3Ks.  The PI:3PS substrate provides a universal PI lipid kinase substrate.

This easy-to-use luminescent assay provides sensitive detection with a high signal-to-background ratios even at low % conversion of substrate. The assay is easily scalable to 1,536-well format, reducing cost per well, and making it well-suited for screening applications.


Lipid Kinase Assay Principle

The lipid kinase reaction is performed by incubating lipid substrate (PI:3PS or PIP2:3PS) with a recombinant enzyme and ATP,  and kinase activity is measured using the ADP-Glo™ Kinase Assay. 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, which is converted to a luminescent signal using a coupled luciferase/luciferin reaction. 

10262MA

Lipid Kinase Assay Accessory Products

The PI3K-Glo™ Class I Profiling Kit contains purified kinases, substrates and ADP-Glo™ Kinase Assay reagents. ADP-Glo™ Kinase assay and substrates, and individual lipid kinases are available for purchase separately.

PI3K Recombinant Enzymes Supplied in the PI3K-Glo™ Class I Profiling Kit

Sources

PI3K (p110α/p85α), 20μg: Complex of recombinant N-terminal FLAG-tagged full-length human p110α (GenBank® Accession Number U79143) and human p85α (no tag; GenBank® Accession Number XM_043865). Co-expressed in Sf9 insect cells.

PI3K (p110α[E545K]/p85α), 20μg:
Complex of recombinant N-terminal FLAG-tagged full-length human p110α (GenBank® Accession Number U79143) and human p85α (no tag; GenBank® Accession Number XM_043865). Co-expressed in Sf9 insect cells.

PI3K (p110α[H1047R]/p85α), 20μg:
Complex of recombinant N-terminal FLAG-tagged full-length human p110α (GenBank® Accession Number U79143) and human p85α (no tag; GenBank® Accession Number XM_043865). Co-expressed in Sf9 insect cells.

PI3K (p110β/p85α), 20μg: Complex of recombinant N-terminal FLAG-tagged full-length human p110β (GenBank® Accession Number NM_006219) and human p85α (no tag; GenBank® Accession Number XM_043865). Co-expressed in Sf9 insect cells.

PI3K (p120γ), 20μg:
Recombinant N-terminal His-tagged full-length human p120γ (GenBank® Accession Number NM _02649) expressed in Sf9 insect cells.

PI3K (p110δ/p85α), 20μg:
Complex of recombinant N-terminal GST-tagged full-length human p110δ (GenBank® Accession Number NM_005026) and human p85α (no tag; GenBank® Accession Number XM_043865). Co-expressed in Sf9 insect cells.
Lipid Kinase Assay Accessory Products
Lipid Kinase Assay ReagentCat. #
ADP-Glo™ Kinase Assay with PI:3PS or PIP2:3PS Substrate
PI:3PS or PIP2:3PS Substrates
Recombinant Human PI3K Enzymes

Specifications

What's in the box?

Item Part # SizeAvailable Separately

ADP-Glo™ Reagent

V912A 1 × 5ml

Kinase Detection Buffer

V913A 1 × 10ml

Kinase Detection Substrate

V914A 1 × 1 each

Ultra Pure ATP, 10mM

V915A 1 × 500μl

ADP, 10mM

V916A 1 × 500μl
PI3K Reaction Buffer, 5X V169A 2 × 1ml

PI3K (p110α/p85α), 5µg

V172A 1 × 50μl View Product
PI3K (p110β/p85α), 5µg V175A 1 × 50μl View Product
PI3K (p120γ), 5µg V176A 1 × 50μl View Product
PI3K (p110δ/p85α), 5µg V177A 1 × 50μl View Product
PIP2:3PS Substrate, 1mg/ml PIP2 V170A 1 × 0.25ml View Product
Lipid Dilution Buffer, 10X V180A 1 × 1ml

MgCl2, 1M

V330A 1 × 1ml

SDS

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Certificate of Analysis

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Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

AA

See Technical Manual for detailed storage recommendations.

Patents and Disclaimers

European Pat. No. 1131441 and Japanese Pat. No. 4520084.

U.S. Pat. Nos. 7,083,911, 7,452,663 and 7,732,128 and other patents.

U.S. Pat. No. 7,700,310 and other patents and patents pending.

U.S. Pat. Nos. 7,741,067, 8,361,739 and 8,603,767 and other patents and patents pending.

U.S. Pat. No. 8,183,007 and other patents and patents pending.

Licensed from Lonza Nottingham Ltd. under U.S. Pat. Nos. 6,599,711 and 6,911,319 and other pending and issued patents.

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