2013 Promega catalogue

Life
Science
Catalog
2013
Worldwide Contact List
Cell Signaling
Protein Kinases and Kinase Assays
Lipid Kinase Assays and Reagents
Product Size Cat.# Price ($)
PI3K-Glo Class I Profiling Kit 1 each V1690 1484
ADP-Glo Kinase Assay with PI:3PS 1,000 assays V1781 1026
10,000 assays V1782 Pls. Enq.
ADP-Glo Kinase Assay with PIP2:3PS 1,000 assays V1791 1026
10,000 assays V1792 Pls. Enq.
Available Separately Size Cat.# Price ($)
PI3K (p110α/p85α), 20µg 200 μl V1721 495
PI3K (p110α[E545K]/p85α), 20µg 200 μl V1731 495
PI3K (p110α[H1047R]/p85α), 20µg 200 μl V1741 495
PI3K (p110β/p85α), 20µg 200 μl V1751 495
PI3K (p120γ), 20µg 200 μl V1761 495
PI3K (p110δ/p85α), 20µg 200 μl V1771 495
PIP2:3PS Lipid Kinase Substrate, 0.25mg 0.25 ml V1701 354
PI:3PS Lipid Kinase Substrate, 0.5mg 0.5 ml V1711 354
For Research Use Only. Not for Use in Diagnostic Procedures.
Description: Phosphatidylinositol (PI) and its phosphorylated derivates, collectively
called phosphoinositides, serve as critical second messengers and
also function in 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 they can be purchased as part of
the PI3K-Glo Class I Profiling Kit, which 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. 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.
Assay Principle: 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 ATPdepletion
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.
Features:
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.
Storage Conditions: Recombinant PI3K Enzymes: Store recombinant
PI3K enzymes below –65°C. Lipid Substrates: Store lipid substrates below
–65°C. Buffers: Store 5X PI3K Reaction Buffer, 10X Lipid Dilution Buffer and
1M MgCl 2 at –30°C to –10°C. ADP-Glo Kinase Assay: Upon receiving
ADP-Glo Kinase Assay, remove ATP and store it below –65°C. Store the rest
of the components at –30 to –10°C.
Protocol
ADP-Glo Lipid Kinase Assay
Part#
TM365
ATP
ATP remaining
P P P
P P P
+ +
ADP
P P
+
PI3K
Step 1. Add ADP-Glo Reagent.
ATP
P P P
ATP remaining
after your reaction.
Deplete
ATP r
P P P
P
PI Substrate
P
PI3P
Step 2. Add Kinase Detection Reagent.
3
P
ADP
P
P
Convert
ATP
Detect
Luciferase/Luciferin
P P P
Newly synthesized ATP.
Light
10262MA
Lipid Kinase Reaction
ADP-Glo Assay
Principle of the ADP-Glo Lipid Kinase Assay.
Section
Contents
Table of
Contents
64
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