2012 Promega catalogue

Cell Signaling
Protease Assays
Protease-Glo Assay
Product Size Cat.# Price ($)
Protease-Glo Assay 1 each G9451 Pls. Enq.
Available Separately
pGloSensor-10F Linear Vector 1 µg G9461 Pls. Enq.
For Research Use Only. Not for Use in Diagnostic Procedures.
Description: The Protease-Glo Assay is a novel method to detect and
measure protease activities using a genetically engineered firefly (Photinus
pyralis) luciferase and represents one example of the GloSensor platform
technology. The assay uses a circularly permuted firefly luciferase, the
GloSensor-10F protein, with a protease recognition site as the protease
substrate. This assay system allows rapid generation of protease substrates
through molecular cloning and coupled transcription/translation cell-free
expression, thus enabling the facile evaluation of protease function.
Oligonucleotides encoding a protease recognition sequence are designed and
cloned into the GloSensor-10F gene located on a linearized vector. The
GloSensor protein containing the protease site of interest is then synthesized
in a cell-free protein expression system and subsequently used as a protease
substrate. Cleavage of the protease recognition sequence leads to activation of
the GloSensor protein and light emission. The level of luminescence
correlates to protease activity. The Protease-Glo Assay has the advantage of
a bioluminescent readout, which provides easy quantitation, high sensitivity and
wide dynamic range.
Visit the Protease-Glo Assay Design Tool to see how to generate your
protease recognition site of interest in the pGloSensor-10F Linear Vector and
express the protein using cell-free translation.
Features:
• Flexible: Use with P´ requiring proteases.
• Avoid Fluorescent Background Problems: Physical and chemical
features of luminescence overcome problems due to fluorescence
interference.
• Greater Sensitivity: Ease and dynamic range of luminescence.
• Open Platform System: Create your own recognition substrates.
• Interrogate Sequences: Excellent tool to determine optimal protease
recognition sequences or effects of amino acid substitutions.
• Web Application: Makes proper oligo design fast and easy; simply enter
your amino acid sequence of interest.
See the Protease-Glo Assay Design Tool.
Storage Conditions: Store all components at –20°C, except the TnT ® SP6
High-Yield Wheat Germ Master Mix, which must be stored at –70°C.
Protocol Part#
Protease-Glo Assay Technical Manual TM303
Luminometer Plates
Product Size Cat.# Price ($)
Luminometer Plates 50 plates Z3291 204.00
For Research Use Only. Not for Use in Diagnostic Procedures.
Description: These plates are White 96-Well Cliniplate, Universal Binding,
Flat Bottom, and are multiwell plates recommended for use with the
Protease-Glo Assay. The plates offer excellent optical, binding precision and
are compatible with all common instruments (manufactured by Thermo Fisher
Scientific).
Features:
• Compatible with All Common Instruments: Excellent optical and
binding properties.
Storage Conditions: Store at room temperature in a cool and dry location.
For complete and up-to-date product information visit: www.promega.com/catalog
DUB-Glo Protease Assay
Product Size Cat.# Price ($)
DUB-Glo Protease Assay (DUB/SENP/NEDP) 10 ml G6260 653.00
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 Part#
DUB-Glo Protease Assay (DUB/SENP/NEDP) Technical Manual TM319
141
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