2013 Promega catalogue

Cell Signaling
ProFluor ® PKA Assay
Product Size Cat.# Price ($)
ProFluor ® PKA Assay 4 plate V1240 1197
For Research Use Only. Not for Use in Diagnostic Procedures.
8 plate V1241 2061
Description: The ProFluor ® PKA Assay measures protein kinase A activity
using purified kinase in a multiwell plate format and involves “add-mix-read”
steps only—ideal for high-throughput applications. The assay begins with a
standard kinase reaction performed with a provided PKA bisamide rhodamine
110 peptide substrate. Following the kinase reaction, a termination buffer
containing a protease reagent is added, which simultaneously stops the kinase
reaction and removes amino acids specifically from the nonphosphorylated
PKA substrate, liberating highly fluorescent rhodamine 110. Phosphorylated
PKA substrate, however, is resistant to digestion by the protease reagent and
remains nonfluorescent. Thus, fluorescence intensity measured in this assay is
inversely correlated with kinase activity. The assay produces excellent Z´-factor
values (>0.8) in either 96- or 384-well plate formats and easily distinguishes
known PKA inhibitors from other compounds.
Features:
• Achieve Highly Predictive Results: Robust Z´ values greater than 0.7 in
either 96- or 384-well plate formats.
• Observe Minimal Test Compound Interference: Rhodamine 110 fluorescent
signal produced is much higher than the fluorescent signal given
off by test compounds.
• Homogeneous: Add-mix-read format reduces the number of steps.
• Non-Radioactive: No radioactive waste disposal and safety issues.
• Choose Your Configuration: Learn more about our custom options for
this product at: www.promega.com/myway/
Storage Conditions: Store the entire system at –20°C. Protect the PKA
R110 Substrate from light. For best results, make solutions fresh and use immediately.
System components should be thawed on ice and returned to –20°C
as soon as possible. The PKA R110 Substrate is provided in 100% DMSO and
therefore requires thawing at room temperature.
Protocol
ProFluor ® PKA Assay Technical Bulletin
Nonphosphorylated Substrate
Phosphorylated Substrate
Part#
TB315
ProFluor ® Src-Family Kinase Assay
Product Size Cat.# Price ($)
ProFluor ® Src-Family Kinase Assay 4 plate V1270 1197
8 plate V1271 2061
For Research Use Only. Not for Use in Diagnostic Procedures.
Description: The ProFluor ® Src-Family Kinase Assay measures the activity
of purified Src-family tyrosine kinases (Src, Lck, Lyn, Fyn, and Hck tested) in
a multiwell plate format and involves “add-mix-read” steps only—ideal for
high-throughput applications. The assay begins with a standard kinase reaction
performed with a provided Src-family kinase bisamide rhodamine 110 peptide
substrate. Following the kinase reaction, a termination buffer containing a
protease reagent is added, which simultaneously stops the kinase reaction and
removes amino acids specifically from the nonphosphorylated substrate, liberating
highly fluorescent rhodamine 110. Phosphorylated substrate, however,
is resistant to digestion by the protease reagent and remains nonfluorescent.
Thus, fluorescence intensity measured in this assay is inversely correlated with
kinase activity. A control peptide (AAF-AMC) is included to control for compounds
that may inhibit the protease. The assay produces excellent Z´ values
(>0.7) in either 96- or 384-well plate formats and easily distinguishes known
Src-family kinase inhibitors from other compounds.
Features:
• Achieve Highly Predictive Results: Robust Z´ values greater than 0.7 in
either 96- or 384-well plate formats.
• Observe Minimal Test Compound Interference: Rhodamine 110 fluorescent
signal produced is much higher than the fluorescent signal given
off by test compounds.
• Control Peptide Included: Use AAF-AMC control peptide to monitor
protease activity and reduce false-positive hits.
• Homogeneous: Add-mix-read format reduces the number of platehandling
steps.
• Non-Radioactive: No radioactive waste disposal and safety issues.
Storage Conditions: For long-term storage, store the system at –20°C.
Protect the Src-Family Kinase R110 Substrate and Control AMC Substrate from
light. Avoid multiple freeze-thaw cycles or exposure to frequent temperature
changes. These fluctuations can greatly alter product stability.
Protocol
ProFluor ® Src-Family Kinase Assay Technical Bulletin
Part#
TB331
4
Cell Signaling
R110
+ Protease
R110
+ Protease
Fluorescent
Nonfluorescent
Fluorescence (RFU)
100,000
90,000
80,000
70,000
60,000
50,000
40,000
30,000
20,000
10,000
0
0 10 20 30 40 50 60 70 80 90 100
Percent Phosphorylated Peptide
3876MD
Schematic and graph demonstrating that the presence of a
phosphorylated amino acid (black circles) blocks the removal of
amino acids from the PKA peptide substrate by the protease.
Section
Contents
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