Shop All Enzyme Detection & Activity Assays

EnzChek™ Protease Assay Kit, green fluorescence, 100-1000 assays (Invitrogen™)

The EnzChek® Protease Assay Kit, green fluorescence, is a fast, simple, and direct fluorescence-based assay for detecting metallo-, serine, acid, and sulfhydryl proteases. The accompanying increase in fluorescence, which can be measured with fluorescence microplate reader, is proportional to protease activity.

See our complete line of Fluorescence Microplate assays.

• Generalized mechanism allows for measurement of a variety of proteases
• Can be used to continuously measure protease kinetics
• Excitation/emission of 505/513 nm allows use of standard fluorescein (FITC) filter settings

This EnzChek® Protease Assay Kit contains a casein derivative that has been extensively labeled with the pH-insensitive, green-fluorescent BODIPY® FL dye, which results in a quenching of the fluorescent dye. Protease-catalyzed hydrolysis releases the highly-fluorescent BODIPY® FL dye-labeled peptides, allowing for quantitative detection of protease activity in solution. The green-fluorescent BODIPY® FL dye has excitation and emission spectra similar to those of fluorescein.

DetectaGene™ Green CMFDG lacZ Gene Expression Kit (Invitrogen™)

The DetectaGene™ Green lacZ Gene Expression Kit includes a fluorescein-based β-galactose derivative that has been chemically modified to include a mildly thiol-reactive chloromethyl group. Once inside a cell, the DetectaGene™ substrate undergoes two reactions: 1) its two galactose moieties are cleaved by intracellular β-galactosidase and 2) either simultaneously or sequentially, its chloromethyl moiety reacts with glutathione and possibly other intracellular thiols to form a membrane-impermeant, peptide-fluorescent dye adduct.

Active Rac1 Pull-Down and Detection Kit (Thermo Scientific™)

The Thermo Scientific Active Rac1 Pull-Down and Detection Kit is a complete kit for the selective enrichment and detection of GTP-bound Rac1 GTPase through specific protein interaction with the Pak1 protein-binding domain.

The Active Rac1 Pull-Down and Detection Kit includes purified GST-Pak1 protein-binding domain (PBD), glutathione agarose resin, positive and negative controls (GTPγS and GDP, respectively), lysis/binding/wash buffer, anti-Rac1 primary antibody, SDS sample buffer, spin columns and collection tubes. The kit was validated using lysates from NIH3T3 cells, a cell line that is known to have robust Rac1 activity.

Features of the Active Rac1 Pull-Down and Detection Kit:

Highly sensitive and accurate—optimized reagents, specific anti-Rac1 antibody and Western blot procedure ensure accurate controls and semi-quantitative results
Validated—functionally tested for Rac1 detection to ensure quality and performance
Compatible—effective with a variety of cell types from mouse, rat and human sources

Applications:
• Follow activation of Rac1 GTPase during cell differentiation, migration, division and cytoskeletal rearrangement
• Study Rac1 dependent lamellipodia formation
• Study the role of active Rac1 in cancer and angiogenesis
• Monitor Rac1 activity after stimulation with growth factors
• Screen small molecule inhibitors for their effect on Rac1 activity

The Active Rac1 Pull-Down and Detection Kit was validated for function and specificity of the active Rac1 enrichment method using cell lysates treated with GTPγS to activate endogenous Rac1 and compared to lysates treated with GDP to inactivate the small GTPase. GTPγS treatment traps Rac1 in the GTP-bound, active form, resulting in a strong signal when endogenous Rac1 is present. GDP treatment pushes Rac1 into the GDP-bound, inactive state, resulting in minimal or no signal, regardless of Rac1 protein levels. The kit is optimized for Western blot detection with an HRP-conjugated secondary antibody (Goat Anti-mouse IgG, Part No. 31430) and Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080). The kit contains sufficient components for 30 pull-down assays.

Rac1 Background:
Rho family GTPases serve many cellular functions, including cell signaling, transcriptional regulation and organization of the actin cytoskeleton. This family of GTPases comprise Rho (RhoA, RhoB, and RhoC), Rac (Rac1, Rac2, Rac3, and RhoG), Cdc42 (Cdc42 and G25K), Rnd (Rnd1, Rnd2, and RhoE/Rnd3), RhoBTB family and the Miro family. These GTPases enable signal transduction from the plasma membrane to the cytosol through GPCR, tyrosine kinase, cytokine and adhesion receptors. Attachment to the plasma membrane is accomplished through geranylgeranyl lipid modifications at the carboxy-terminus of the protein. Rac1 activation results in actin polymerization and appears as membrane ruffling at the cellular periphery. Rac1 activation also results in lamelipodia formation.

The Rac1 GTPase transduces signals through tyrosine kinases, adhesion molecules or cytokine/chemokine receptors after stimulation with growth factors (EGF, insulin, PDGF, NGF), integrins (fibronectin) or chemoattractants (fMLP). For example, stimulation with EGF results in PI3 kinase activation, resulting in cell growth and reorganization at the cell periphery (membrane ruffling). Alternatively, tyrosine receptor kinase signaling through Rac1 leads to activation of the MAPK stress response pathways SAPK (JNK) and p38. Stimulation of cells with fibronectin results in integrin-mediated cell spreading. Two of the main effector proteins of Rac1 are Pak1 and phosphoinositol 4-phosphate 5-kinase. Pak1 (p65 Pak) is a kinase that activates the JNK pathway, while phosphoinositol 4-phosphate 5-kinase promotes actin filament assembly. Rac is critical for T-cell development and for promoting differentiating cells. However, the Rho family of GTPases can work agonistically during cell signaling and and antagonistically during differentiation.

More Product Data
Measure activation of small GTPases via their specific downstream effectors
Detection and localization of active GTPases in neuronal cell differentiation

EnzChek™ Protease Assay Kit, red fluorescence, 100-1000 assays (Invitrogen™)

The EnzChek® Protease Assay Kit, red fluorescence, is a fast, simple, and direct fluorescence-based assay for detecting metallo-, serine, acid, and sulfhydryl proteases. The accompanying increase in fluorescence, which can be measured with a fluorescence microplate reader, is proportional to protease activity.

See our complete line of Fluorescence Microplate assays.

• Generalized mechanism allows for measurement of a variety of proteases
• Can be used to continuously measure protease kinetics
• Excitation/emission similar to Texas Red® dye (589/617 nm)

This EnzChek® Protease Assay Kit contains a casein derivative that has been extensively labeled with the red-fluorescent BODIPY® TR-X dye, which results in a quenching of the fluorescent dye. Protease-catalyzed hydrolysis releases the highly-fluorescent BODIPY® TR-X dye-labeled peptides, allowing for quantitative detection of protease activity in solution. The red-fluorescent BODIPY® TR-X dye has excitation and emission spectra similar to those of the Texas Red® fluorophore.

Pierce™ Kinase Enrichment Kit with ATP Probe (Thermo Scientific™)

This Thermo Scientific Pierce Kinase Enrichment Kit utilizes an ActivX™ ATP Probe to covalently label the active site of ATPases, including chaperones and metabolic enzymes, to enable their selective enrichment using a desthiobiotin tag.

ActivX ATP Probes feature an amine-reactive nucleotide analog and a desthiobiotin (biotin analog) tag that facilitates selective labeling of lysines in the kinase active site and then subsequent enrichment and recovery of labeled protein. These features allow identification and profiling of target enzyme classes across samples or assessment of the specificity and affinity of enzyme inhibitors.

Features of the Pierce Kinase Enrichment Kit:

Specific – label only the conserved active-site lysines of nucleotide-binding proteins
Flexible – use for in vitro labeling of ATPase enzymes derived from cells or tissues
Compatible – use with Western blot or mass spectrometry (MS) workflows

Applications:
• Profile small-molecule binding affinities and active-site inhibition in a dose-dependent manner
• Identify dozens to hundreds of inhibitor targets and off-targets from tissues, cells and subcellular proteomes
• Enrichment of enzymes based on function

Thermo Scientific ActivX Desthiobiotin-ATP is a nucleotide derivative that covalently modifies the active site of enzymes at conserved lysine residues in the nucleotide binding site. The structure of these probes consists of a modified biotin (desthiobiotin) attached to the nucleotide through a labile acyl-phosphate bond. Desthiobiotin is a biotin analog that binds less tightly to biotin-binding proteins resulting in binding that is easily reversed by biotin displacement, low pH or heat denaturation.

Desthiobiotin-ATP probes can be used to selectively enrich, identify and profile target enzyme classes or assess the specificity of enzyme inhibitors. Because many ATPases and other nucleotide-binding proteins bind nucleotides or inhibitors even when they are enzymatically inactive, the desthiobiotin probes allow profiling of both inactive and active enzymes in a complex sample. Preincubation of samples with small-molecule inhibitors that compete for active sites can be used to determine inhibitor binding affinity. Active-site nucleotide probes also can be used to identify inhibitor off-targets.

These products are subject to a limited use label license.

Related Products
ActivX™ Desthiobiotin-ATP Probe
Pierce™ Kinase Enrichment Kit with ADP Probe
ActivX™ Desthiobiotin-ADP Probe

Pierce™ Renilla-Firefly Luciferase Dual Assay Kit (Thermo Scientific™)

The Thermo Scientific Pierce Renilla-Firefly Luciferase Dual Assay Kit provides the necessary reagents to simultaneously detect intracellular Renilla and Red Firefly luciferase activity in mammalian whole cell lysates.

Features of the Renilla-Firefly Luciferase Dual Assay Kit:

Simultaneous—concurrent, filter-based, wavelength-separated detection of two luciferase activities
Sensitive—measure Green Renilla and red firefly luciferase activities in the same sample
Fast—no quenching step required, unlike in traditional sequential dual assays
Multiplex—capable of quantitating two cellular activities in the same sample(s)

The Pierce Renilla-Firefly Luciferase Dual Assay Kit is a highly sensitive assay that allows for the simultaneous detection of Green Renilla and Red Firefly luciferase activity. Green Renilla luciferase acts as an experimental reporter with constitutively active red firefly as a normalization control. As shown in the animation video below, this reporter-and-control combination enables simultaneous monitoring of experimental reporter and control luciferase activities in a single-read assay without the need for two-step addition of substrate reagents or quenching. The assay working solution contains substrates for both luciferases, and the reactions occur simultaneously with flash-type kinetics. The resulting luminescent signals are spectrally resolvable using filters. In a single sample, researchers can assay transcriptional activity of regulatory elements, signal transduction pathways, and effects of activators or inhibitors.

Includes:
Cell lysis buffer, reaction buffer, and substrates coelenterazine and D-luciferin

Requires:
Green Renilla and Red Firefly luciferase reporters; Filter sets at 525nm±20nm for Green Renilla Luc and 615 to 675nm for Red Firefly Luc. Luminometer or other instrument capable of monitoring luminescence, such as Thermo Scientific Luminoskan Ascent and Varioskan Flash Microplate Readers; Injector required for assessing more than 24 wells at a time.

Applications:
• Study two regulatory elements at the same time
• Monitor two signaling pathways simultaneously
• Enable studying more than one target per screen (e.g. off-target effects)

Green Renilla produces brighter bioluminescent signal than firefly and native Renilla luciferases. The bioluminescent signal (λmax= 535nm) produced by Green Renilla luciferase protein results from the oxidation of coelenterazine. The second assay uses red firefly luciferase, which is a mutant form of the Japanese firefly luciferase Luciola cruciata. This luciferase produces a red-shifted emission spectrum (λmax= 613nm) that results from the oxidation of D-luciferin in the presence of ATP.

More Product Data
Simultaneous dual-emission detection of luciferase reporter assays

Related Products
Pierce™ Renilla-Firefly Luciferase Dual Assay Kit

NA-Fluor™ Influenza Neuraminidase Assay Kit (Invitrogen™)

The NA-Fluor™ Influenza Neuraminidase Assay Kit provides validated reagents and a standardized protocol for conducting neuraminidase enzyme assays, including neuraminidase inhibitor (NI) susceptibility screening using a fluorescent MUNANA substrate.

Key product features:
• Efficient design—neuraminidase assay in one complete kit
• Optimized formulations—assay reagents have been optimized for maximum performance based on NISN protocols
• Robust application—functional detection of NI-resistant virus in mixed viral populations
• Easy of use & flexible screening—stable fluorescent signal enables batch-mode or high throughput processing

Neuraminidase Assasy in One Complete Kit
The NA-Fluor™ Influenza Neuraminidase Assay Kit includes comprehensive protocols for titering viral isolates based upon neuraminidase activity and conducting neuraminidase enzyme inhibition assays. The assay can also be used for monitoring neuraminidase enzyme activity from non-viral or bacterial sources. The NA-Fluor™ Kit contains reagents for 10 96-well microplates sufficient for a total of 960 assay wells and includes the following components: fluorescent MUNANA neuraminidase substrate, assay buffer, and stop solution to enhance and stabilize signal. The assay is performed in standard black microplates (microplates not provided in kit) and is read on standard fluorometers.

Neuraminidase Assay Compares to NISN Protocols
The NA-Fluor™ assay reagent formulations are optimized to be comparable to several well-established MUNANA-based assay protocols, such as:
• The MUNANA substrate concentration, assay buffer formulation and assay conditions are consistent with NISN IC-50 determination protocols
• Data generated using the NA-Fluor™ assay corresponds to data generated with established MUNANA based protocols
These key parameters enable investigators to compare data acquired during current NI resistance surveillance screens using the NA-Fluor™ assay to data acquired using their previous MUNANA assay protocols (Figure 1).

Detection of NI-Resistant Virus in Mixed Viral Populations
The large shift in IC-50 values between sensitive and oseltamivir-resistant virus using the NA-Fluor™ assay enables detection of mutant virus in mixed viral samples (Figure 2). This capability is critical for identifying resistant virus in clinical isolates presenting mixed populations of resistant and sensitive virus during NI susceptibility surveillance.

Flexibility for Screening Neuraminidase Activity
The NA-Fluor™ assay provides an easy, flexible format for the screening of several to several hundred viral isolates, or the screening of thousands of compounds during high-throughput lead discovery with quality data at high confidence levels. With superior performance, the assay has demonstrated a Z´ of 0.78-0.8, making it strongly capable for use in high-throughput screening. The fluorescent reaction product remains stable for hours at room temperature after the assay is complete, enabling read-time flexibility and comparable data from first plate to last. The assay signal remains nearly constant and IC-50 values (data not shown) are identical from data collected up to 4 hours at room temperature and up to 4 days at 4 °C after assay termination (Figure 3). The NA-Fluor™ assay has been optimized as an end-point assay run at 37 °C for one hour following NI pre-incubation. However, the rate of MUNANA substrate turnover remains linear for more than 2 hours with viral neuraminidase, allowing the assay to be performed for as little as 20 minutes to save time or for as long as 2 hours to increase signal output. The assay can also be conducted in real-time without the addition of stop solution for those investigators who want to perform their own assay development or monitor rates of substrate turnover in the presence of inhibitor (Figure 4).

For Research Use Only. Not for use in diagnostics procedures.

ActivX™ Desthiobiotin-ATP Probe (Thermo Scientific™)

Individual ActivX Desthiobiotin-ATP Probe, 16 × 12.6µg
Molecular Weight: 1259.48
For use with the Pierce Kinase Enrichment kits.

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Pierce™ Kinase Enrichment Kit with ATP Probe
Pierce™ Kinase Enrichment Kit with ADP Probe
ActivX™ Desthiobiotin-ADP Probe

KDalert™ GAPDH Assay Kit (Invitrogen™)

The Ambion® KDalert™ GAPDH Assay Kit is for the reliable measure of GAPDH enzyme activity in cultured human, mouse, or rat cells in less than 30 minutes using a microplate fluorometer. The kit includes sufficient reagents for 375 reactions.

• Assess GAPDH siRNA delivery in 1/3 the time for 1/3 the cost of real-time PCR
• Analyze 1-96 samples simultaneously
• Measure both GAPDH siRNA-induced knockdown AND transfection-induced toxicity
• Compatible with a wide variety of cells and a broad range of culture conditions

The KDalert GAPDH Assay Kit is an ideal positive control for transfection optimization experiments and also measures transfection induced cytoxicity. It is designed for use with Ambion® Silencer® GAPDH siRNA.

Rapid, Time-Saving Procedure
Use the assay to optimize siRNA transfection by transfecting individual cell samples with a GAPDH siRNA and a negative control siRNA. Two to three days after transfection, simply add the included cell lysis buffer to the cells, incubate for 20 minutes, add the diluted master mix of assay reagents, and read the increase in fluorescence four minutes later using a microplate or standard fluorometer. The assay procedure can be completed in about 30 minutes with minimal sample handling.

One Assay for Two Readouts
Because GAPDH is expressed at relatively constant levels, the assay can also be used to monitor transfection agent induced toxicity. For this analysis, GAPDH enzyme activity from negative control siRNA-transfected cells is compared to that of untreated cells. Reduced GAPDH activity in negative control-transfected cells compared to non-transfected cells is an indication that the transfection-induced cytotoxicity.

Accessory Products:
The KDalert™ Kit is designed for use with Silencer® GAPDH siRNAs (SKUs #AM4605, AM4633, AM4634, AM4624, AM4632, or AM4631). Additional KDalert™ Lysis Buffer (SKU #AM8790G) is also available separately.

Gal-Screen™ β-Galactosidase Reporter Gene Assay System for Mammalian Cells (Invitrogen™)

The Gal-Screen® assay system combines direct cell lysis with rapid, ultra-sensitive chemiluminescent detection of β-galactosidase reporter enzyme.

• Homogeneous assay format allows detection of β-galactosidase in the presence of normal culture media without removal of media and without an additional cell lysis step, providing the easiest and most streamlined assay procedure possible.
• The kinetics of the glow-assay provides a "window" during which measurements may be performed, facilitating HTS applications where assay automation is used.
• Can be used with either mammalian or yeast model systems, providing flexibility in choice of model systems.
• Wide dynamic range of β-galactosidase assay enables accurate measurement of enzyme from femtogram to nanogram range.
• Assay sensitivity is 100 to 1,000-fold better than either the isotopic⁄non-isotopic assays for chloramphenicol acetyl transferase (CAT) or the colorimetric⁄fluorescent assays for β-galactosidase, providing greater sensitivity than competing assay technologies.
• Highly sensitive assay with a wide dynamic range permits detection of high and low levels of reporter without performing numerous sample dilutions.
• Non-radioactive reporter gene assay kit eliminates concerns over use of radioisotopes.
• Assay can be completed in about one hour, providing fast assay turnaround.

Ideal for Screening
This homogeneous assay is ideally suited for screening applications where assay automation is required. The Gal-Screen® system uses Galacton-Star® chemiluminescent substrate for convenient measurement in a luminometer. Light emission reaches maximum in 60-90 minutes and remains constant for 45-90 minutes.

For Research Use Only. Not for use in diagnostics procedures.

Active Cdc42 Pull-Down and Detection Kit (Thermo Scientific™)

The Thermo Scientific Active Cdc42 Pull-Down and Detection Kit is a complete kit for selective enrichment and detection of GTP-bound Cdc42 GTPase through specific protein interaction with the Pak1 protein-binding domain.

The Active Cdc42 Pull-Down and Detection Kit includes purified GST-Pak1 protein-binding domain (PBD), glutathione agarose resin, positive and negative controls (GTPγS and GDP, respectively), lysis/binding/wash buffer, anti-Cdc42 primary antibody, sample buffer, spin columns and collection tubes. The kit was validated using lysates from NIH 3T3 cells, a cell line that is known to have robust Cdc42 activity.

Features of the Active Cdc42 Pull-Down and Detection Kit:

Highly sensitive and accurate—optimized reagents, specific anti-Cdc42 antibody and Western blot procedure ensure accurate controls and semi-quantitative results
Validated—functionally tested for Cdc42 detection to ensure quality and performance
Compatible—effective with a variety of cell types from mouse, rat and human sources

Applications:
• Follow activation of Cdc42 GTPase during cell differentiation, migration, division, and cytoskeletal rearrangement
• Study the activation of Cdc42 during filopodia formation
• Monitor Cdc42 activity after stimulation with growth factors
• Screen small molecule inhibitors for their effects on Cdc42 activity

The Active Cdc42 Pull-Down and Detection Kit was validated for the function and specificity of the active Cdc42 enrichment method using cell lysates treated with GTPγS to activate endogenous Cdc42 and compared to lysates treated with GDP to inactivate the small GTPase. GTPγS treatment traps Cdc42 in the GTP-bound, active form, resulting in a strong signal when endogenous Cdc42 is present. GDP treatment pushes Cdc42 into the GDP-bound, inactive state, resulting in minimal or no signal regardless of Cdc42 protein levels. This kit is optimized for Western blot detection with an HRP-conjugated secondary antibody (Goat Anti-mouse IgG, Part No. 31430) and Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080). The kit contains sufficient components for 30 pull-down assays.

Cdc42 Background:
Rho family GTPases serve many cellular functions, including cell signaling, transcriptional regulation and organization of the actin cytoskeleton. This family of GTPases comprise Rho (RhoA, RhoB, and RhoC), Rac (Rac1, Rac2, Rac3, and RhoG), Cdc42 (Cdc42 and G25K), Rnd (Rnd1, Rnd2, and RhoE/Rnd3), RhoBTB family and the Miro family. These GTPases enable signal transduction from the plasma membrane to the cytosol through GPCR, tyrosine kinase, cytokine and adhesion receptors. Attachment to the plasma membrane is accomplished through geranylgeranyl lipid modifications at the carboxy-terminus of the protein. Cdc42 activation results in the polymerization of actin filaments and filopodia formation.

The Cdc42 subfamily of RhoGTPase has been less well-characterized than the Rho and Rac families. Both Cdc42 and Rac1 are activated through tyrosine receptor kinase signaling leading to SAPK and p38 stress kinase pathway activation. Cdc42 is also activated by the chemoattractant fMLP in neutrophils. Fibronectin activates Cdc42 and Rac1 to induce cell spreading, and stimulation with TNF-alpha and IL-1 results in changes in the actin cytoskeleton. There is significant cross-talk between Cdc42 and Rac1, as they act in overlapping pathways, and in some cases, Cdc42 may act upstream of Rac1 during signal transduction. Some of the main effector proteins of Cdc42 are Pak1, N-WASP and IQGAP. Pak1 is a kinase involved in the activation of JNK in the SAPK stress pathway. N-WASP is an effector that induces filopodia formation, and IQGAP interacts with F-actin filaments. In differentiating neurons, Cdc42 plays an active role in neurite outgrowth. However, the Rho family of GTPases can work agonistically during cell signaling and antagonistically during differentiation.

More Product Data
Measure activation of small GTPases via their specific downstream effectors

PKA (Protein Kinase A) Colorimetric Activity Kit (Invitrogen™)

The PKA Activity research-use-only kit is a colorimetric activity assay designed for the quantification and detection of protein kinase A activity in cell lysates, tissue extracts and buffer samples.

This complete, ready-to-use kit includes a PKA substrate-coated 96-well plate(s), PKA standard (5,000 U), phospho-PKA substrate antibody, and other components to perform the assay. A 96-well microplate reader capable of reading optical density at 450 and 650 nm is required for use of this kit.

Performance characteristics
• Assay type: colorimetric activity kit
• Sample types: cell lysates, tissue extracts, buffer samples
• Sensitivity: 0.366 U/mL
• Standard curve range: 5 U/mL–25 U/mL
• Reactivity: species independent

Background
PKA is a member of an important class of kinases, referred to as Arg-directed kinases or AGC-family kinases, that includes cAMP-dependent protein kinase (PKA or cAPK), cGMP-dependent protein kinase (PKG), protein kinase C, Akt, and RSK. These kinases share a substrate specificity characterized by Arg at position 3 relative to the phosphorylated serine or threonine. The second messenger cyclic AMP (cAMP) activates PKA in mammalian cells and controls many cellular mechanisms such as gene transcription, ion transport, and protein phosphorylation2. Inactive PKA is a heterotetramer composed of a regulatory subunit (R) dimer and a catalytic subunit (C) dimer. In this inactive state, the pseudosubstrate sequences on the R subunits block the active sites on the C subunits. PKA shares substrate specificity with Akt (PKB) and PKC. Substrates that present this consensus sequence and are phosphorylated by PKA are Bad (Ser155), CREB (Ser133), and GSK-3.

PKA has been implicated in numerous cellular processes, including modulation of other protein kinases, regulation of intracellular calcium concentration, and regulation of transcription. Transcriptional responses to increased cAMP occur through activation of the cAMP response element–binding protein (CREB), cAMP response element modulator (CREM), and activating transcription factor 1 (ATF1). Each of these transcription factors contains a kinase-inducible domain containing a conserved site for phosphorylation by PKA.

Assay principle
The PKA activity kit is designed to quantitatively measure PKA activity in a variety of samples. A recombinant PKA standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve. The kit utilizes an immobilized PKA substrate bound to a microtiter plate. Samples containing PKA will, in the presence of the supplied ATP, phosphorylate the immobilized PKA substrate. After a 90-minute incubation followed by a wash, a rabbit antibody specific for the phospho-PKA substrate binds to the modified immobilized substrate. An antibody specific for rabbit IgG labeled with peroxidase is also added to the plate to bind to the rabbit anti-phospho-PKA substrate. After a short incubation and wash, substrate is added and the intensity of the color developed is directly proportional to the amount of PKA in the samples and standards.

Related links
Learn more about ELISA kits
Learn more about other immunoassays

Z'-LYTE™ Kinase Assay Kit - Ser/Thr 18 Peptide

A coumarin and fluorescein-labeled peptide substrate which can be used for kinase screening in an antibody-free format. This peptide is based upon an optimized synthetic peptide substrate for DYRK1A, and containing an R-P-X-(S⁄T)-P consensus sequence, where X is any residue.

Yeast beta-Galactosidase Assay Kit (Thermo Scientific™)

Thermo Scientific Yeast β-Galactosidase Assay Kit is specially formulated for yeast cells and ideal for identifying protein interactions in-vivo using two-hybrid systems.

Features of Yeast beta-Galactosidase Assay Kit:

• Efficient lysis of yeast cells and a colorimetric detection system
• Quantitative or qualitative assay for yeast colonies grown on solid media or in suspension
• Colorimetric assay easily measured in a plate reader or spectrophotometer (420nm)
• Adaptable to 96-well microplates or tube assay formats
• Allows cells obtained from solid media or liquid culture to be assayed directly with no harvesting or washing steps.• Perform homogeneous screening assays or measure activity secondarily after cell lysis
• Also compatible with prepared lysates from other species such as bacteria

Yeast β-Galactosidase Assay Kit allows qualitative or quantitative determination of β-galactosidase activity in solution directly from colonies growing on solid medium. A portion of the colony is suspended in a mixture of Thermo Scientific Y-PER Yeast Protein Extraction Reagent and β-Galactosidase Assay Buffer. After a brief incubation period, the solution turns yellow from the hydrolysis of o-nitrophenyl-β-D-galactopyranoside (ONPG) to o-nitrophenol (ONP) and galactose in a mildly alkaline solution. The assay becomes quantitative if calibrated to cell number by measuring the optical density at 660nm.

The gene encoding β-galactosidase (lacZ) of E. coli has been widely used as a reporter gene in many different prokaryotic and eukaryotic organisms. β-Galactosidase activity is also commonly used as an indicator of protein-protein interactions in-vivo using two-hybrid systems. The interaction strength is verified or quantitated using a β-galactosidase activity assay.

Pierce™ Cypridina Luciferase Flash Assay Kit (Thermo Scientific™)

The Thermo Scientific Pierce Cypridina Luciferase Flash Assay Kit provides researchers with a highly sensitive assay for transcriptional activity of regulatory elements in mammalian cell culture media and whole cell lysate.

Features of the Cypridina Luciferase Flash Assay Kit:

Sensitive—high sensitivity allows utilization of smaller numbers of cells
Cost effective—sensitive assays result in decreased reagent consumption
Secreted luciferase—allows real-time assays and kinetic studies without destroying cells
Ideal for co-transfection—uses a different substrate than Renilla, Gaussia, or firefly luciferases
Compatible—assay reagents compatible with other Cypridina or Vargula luciferases
Time-saving—assays using secreted luciferases require minimal sample handling
Automation-friendly—amenable to high throughput screens
Convenient—contains a universal cell lysis buffer and optimized flash assay reagent
Safe—allows one to perform non-radioactive assays

This Flash Assay Kit contains reagents for measuring the activity of Cypridina luciferase in mammalian cell culture media and lysates. When used with Thermo Scientific Cypridina Luc Vectors, the kit provides an extremely sensitive bioluminescent reporter assay system for secreted or intracellular detection of promoter or pathway activity. Cypridina luciferase is highly secreted into the cell culture media, allowing for live cell monitoring of reporter activity. The signal produced by Cypridina is considerably greater than signal from either firefly or Renilla luciferases assayed under similar conditions.

Includes:
Cell lysis buffer, reaction buffer and substrate

Requires:
Cypridina luciferase and luminometer or other instrument capable of monitoring luminescence, such as Thermo Scientific Luminoskan Ascent and Varioskan Flash Microplate Readers.

Applications:
• Promoter studies for analyzing cis- regulatory elements and trans-acting factors
• Drug screening
• siRNA and miRNA screening
• Multiplexed assays to study off-target effects
• Secretory pathway/protein localization reporter assays
• Signal transduction pathway analysis
• RNA splicing studies

The Cypridina luciferase is a 61kDa protein from the marine ostracod, Cypridina noctiluca. Cypridina luciferase is highly secreted into the cell culture media, allowing for live cell monitoring of reporter activity. Light output captured using a luminometer can be correlated with the amount of Cypridina luciferase protein produced and used to determine the activity of the promoter driving Cypridina expression. The Cypridina gene in Thermo Scientific Cypridina Luc Vectors is modified for expression in mammalian cells.

The assay kit is optimized for use with our Cypridina Luc Vectors but is fully compatible with any luciferases that use Vargulin as a substrate. Because Cypridina uses a different substrate than Renilla, Gaussia, or firefly luciferases, co-transfection and assays with up to three reporters are possible. The signal produced by Cypridina shows strong flash kinetics and is considerably greater than flash signals from either firefly or Renilla luciferases assayed under similar conditions.

More Product Data
Highly sensitive multiplex luciferase reporter assays
Luciferase assays in hard-to-transfect Jurkat cells
Monitoring neuronal differentiation using multiplexed luciferase reporters
Activation of the antioxidant response pathway by pesticide chemicals
Versatile luciferases: microplate luminometers and flash luciferase assays