2013 Promega catalogue

Cell Signaling
DUB-Glo Protease Assay
Product Size Cat.# Price ($)
DUB-Glo Protease Assay (DUB/SENP/NEDP) 10 ml G6260 667
For Research Use Only. Not for Use in Diagnostic Procedures.
50 ml G6261 Pls. Enq.
Description: The DUB-Glo Protease Assay (DUB/SENP/NEDP) is a
homogeneous, bioluminescent assay that measures the activity of numerous
deconjugating enzymes including deubiquitinating (DUB), deSUMOylating
(SENP) and deneddylating (NEDP) proteases. These proteases reverse the
protein modification by ubiquitin and ubiquitin-like proteins (Ubl proteins) and
thus are integral components in the complex mechanisms of posttranslational
protein regulation in eukaryotes.
Features:
• Greater Sensitivity: The luminescent format provides enough sensitivity
to enable use of a simple peptide-based substrate, Z-RLRGG-aminoluciferin,
for assaying deconjugating proteases. Fluorescence generally requires
the use of full-length substrates.
• Broad Dynamic Range: The assays are linear over 2–3 logs of deconjugating
protease concentrations.
• Signal Stability: The coupled-enzyme format results in very stable signal
with a half-life >3 hours. Substrate depletion is not a concern as it is when
using the full-length substrates, Ub-AMC, SUMO-AMC or Nedd8-AMC.
• Fast: Maximum sensitivity is reached in 10–30 minutes after reagent
addition because the signal is not dependent on accumulation of cleaved
product for sensitivity in the coupled-enzyme format.
• Accurate and Robust: The broad linear range and excellent sensitivity
readily translate to accurate kinetic analysis of inhibitors. Assays can be
scaled to 384-well with suitable Z´ factors.
• Greater Flexibility: The K m values for the peptide substrates are much
higher than they are for full-length substrates, yet the sensitivity of the
luminescent assay allows the assay to be run significantly below K m while
still achieving good signal-to-background ratios for extended time periods.
A single luminescent substrate concentration can be used for a wide
variety of DUB/SENP/NEDP proteases without worrying about substrate
depletion or substrate inhibition.
• Batch-Processing Capability: The homogeneous coupled-enzyme format
results in a continuous signal, providing excellent stability and allowing
plates to be read over an extended period of time.
Storage Conditions: Store components at –20°C protected from light.
Protocol
DUB-Glo Protease Assay (DUB/SENP/NEDP) Technical Manual
Part#
TM319
ApoTox-Glo Triplex Assay
Product Size Cat.# Price ($)
ApoTox-Glo Triplex Assay 10 ml G6320 828
5 × 10 ml G6321 Pls. Enq.
For Laboratory Use.
Description: The ApoTox-Glo Triplex Assay combines three assay chemistries
to easily assess viability, cytotoxicity and apoptosis events in the same
cell-based assay well. First, viability and cytotoxicity are determined by measuring
two differential protease biomarkers simultaneously with the addition of a
single nonlytic reagent containing two peptide substrates. The live-cell protease
activity is restricted to intact viable cells and is measured using a fluorogenic,
cell-permeant peptide substrate (GF-AFC Substrate). The substrate enters
intact cells, where it is cleaved to generate a fluorescent signal proportional
to the number of living cells. This live-cell protease activity marker becomes
inactive upon loss of membrane integrity and leakage into the surrounding
culture medium. A second, cell-impermeant, fluorogenic peptide substrate (bis-
AAF-R110 Substrate) is used simultaneously to measure dead-cell protease
activity that has been released from cells that have lost membrane integrity.
This results in ratiometric, inversely correlated measures of cell viability and
cytotoxicity. The ratio of viable cells to dead cells is independent of cell number
and, therefore, can be used to normalize data. A second reagent containing luminogenic
DEVD-peptide substrate for caspase-3/7 and Ultra-Glo Recombinant
Thermostable Luciferase is added. Caspase-3/7 cleavage of the substrate
releases luciferin, which is a substrate for luciferase and generates light. The
light output, measured with a luminometer, correlates with caspase-3/7 activation
as a key indicator of apoptosis.
Features:
• Measure Viability, Cytotoxicity and Apoptosis in the Same Sample
Well: Determine mechanism of cell death for cells in the same sample
well.
• Easily Implement: Assay follows a simple sequential “add-mix-measure”
format.
• Normalize Data with a Built-In Control: The ratio of the number of live
cells/number of dead cells is independent of cell number and normalizes
data. This normalization makes results more comparable well-to-well,
plate-to-plate and day-to-day.
• Flexible and Easily Automated: The volumes of each assay component
can be scaled to meet throughput needs and is amenable to automation in
96- and 384-well plates.
• Improves Efficiency and Saves on Lab Budget: Reduces cell culture
and labor costs by performing three assays in a single well.
Storage Conditions: Store all components at –20°C protected from light.
Protocol
ApoTox-Glo Triplex Assay Technical Manual
Part#
TM322
8
Epigenetics
Section
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