2012 Promega catalogue

Life
Science
Catalog
2012
Worldwide Contact List
Section
Contents
Table of
Contents
Cell Signaling
62
Nonphosphorylated Substrate
Fluorescence (RFU)
100,000
90,000
80,000
70,000
60,000
50,000
40,000
30,000
20,000
10,000
R110
Fluorescent
+ Protease
Phosphorylated Substrate
R110
Nonfluorescent
0
0 10 20 30 40 50 60 70 80 90 100
Percent Phosphorylated Peptide
+ Protease
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.
ProFluor ® Src-Family Kinase Assay
Product Size Cat.# Price ($)
ProFluor ® Src-Family Kinase Assay 4 plate V1270 1185.00
For Research Use Only. Not for Use in Diagnostic Procedures.
8 plate V1271 2040.00
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: Fluorescent signal
from Rhodamine 110 is much stronger than fluorescents 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 Part#
ProFluor ® Src-Family Kinase Assay Technical Bulletin TB331
3876MD
SAM 2® Biotin Capture Membrane
Product Size Cat.# Price ($)
SAM2® Biotin Capture Membrane 96 samples V2861 400.00
For Laboratory Use.
7.6 × 10.9 cm V7861 416.00
Description: The SAM 2® Biotin Capture Membrane binds biotinylated molecules
based on their affinity for streptavidin. The proprietary process by which
the SAM 2® Membrane is produced results in a high density of streptavidin on
the filter, providing rapid, quantitative substrate binding in the nmol/cm 2 range,
depending upon the substrate used. In addition, the membrane is designed
to minimize nonspecific binding. The membrane is available either as a large,
prenumbered, partially cut sheet (approximately 10.5 × 15.0cm; Cat.# V2861)
or as a smaller, uncut sheet (approximately 7.6 × 10.9cm; Cat.# V7861). The
partially cut membrane allows easy separation into 96 individual squares and is
designed for small-scale experiments where high binding capacity is required.
The uncut sheet can be analyzed as a whole membrane or may be cut to
the size desired. The uncut membrane allows for sample application using a
multichannel pipettor. Both membranes may be analyzed using phosphorimaging
analysis, autoradiography or scintillation counting to quantitate results. The
membranes have also been used successfully with chemiluminescence detection
techniques. The use of fluorescence for detection of captured molecules is
not recommended at this time.
Features:
• Use a Variety of Substrates: Analysis of biotinylated substrates can be
applied to a wide variety of substrate types without the need to optimize
each substrate for binding to a matrix. The user can perform experiments
with a wide array of sample numbers and sizes without changing the
analysis technique, since the membrane is available in 96-square (partially
cut) and solid sheet (uncut) formats.
• Minimize Nonspecific Binding: The combination of protein denaturant
and high-salt washes minimizes nonspecific binding to the membrane
without interfering with the high-affinity interaction between streptavidin
and biotin.
• Obtain High Signal-to-Noise Ratios: The stringent washing conditions
employed assist in attaining very low background counts.
• Perform Kinetic Studies: Membrane can linearly bind biotinylated
substrates up to the nmol/cm 2 range. Allows for kinetic studies.
• Strong Binding Reaction: Membrane retains the biotin conjugate over 8
logs of pH (pH 2–10), changes in temperature, organic solvents, ionic and
nonionic detergents (SDS, CHAPS, Triton ® X-100, Tween ® 20 and Tween ®
80) and denaturing agents (5M guanidine-HCl and 2M urea).
• Rapid: Binds within 1 minute.
• Convenient: Compatible with enzyme assays using radioactive detection.
Membranes manufactured by this method have been shown to allow
chemiluminescent detection.
Storage Conditions: Store membranes at –20°C in resealable bag.
Protocol Part#
SAM2® Biotin Capture Membrane Technical Bulletin TB547
For complete and up-to-date product information visit: www.promega.com/catalog