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Signal Transduction

ACTOne Cell Based Assay Technology

A cell based assay system for studying GPCR and PDE activities in vitro

CodexBiosolutions provides cell based assay products and services to academic and governmental research institutes, and other research labs within the pharmaceutical and biotechnology industries. The company is headquartered in the I-270 Hi-Tech corridor in the state of Maryland. The core products of CodexBiosolution are ACTOne Cell lines and membrane potential kits which are licensed from BD Biosciences. They also develop other new cell based products to meet the demands from various customers.

ACTOne Technology is the key platform used by Codex BioSolutions for development of Cell Based Assay for GPCR and PDE.

It is based upon a modified cyclic nucleotide-gated (CNG) ion channel as a biosensor of cAMP activity in live cells. This channel responds in real-time to increases or decreases in intracellular cAMP levels by coordinately altering cation flux (e.g., calcium, potassium or sodium), which can be measured by calcium-sensitive fluorescent indicators, or by Codex’s optimized membrane-potential dye on broadly-available plate readers or on single-cell imagers. By juxtapositioning this biosensor adjacent to adenylyl cyclase, the enzyme responsible for synthesis of cAMP, in the membrane of cells that also express the GPCR target of interest, the biosensor provides ultrasensitive responses to cAMP fluctuations (Rich et. al., J. Gen. Physiol. 2000;116:147–161). The technology platform enables live-cell GPCR screening or deorphanization of agonists, antagonists and allosteric modulators of Gs, Gi or Gq-coupled receptors in 1536-well formats. Because the technology directly measures cAMP through CNG channels, it detects GPCR activity through its nature pathway. No engineering or modifications to the G protein is required. The assay is highly sensitivity to intracellular cAMP change. It has excellent signal to noise ratio and high z’ value. It is also cost-effective and easy to use. Preliminary evidence has been developed that supports the ability to freeze these cell lines in ready-to-screen format, and for enabling receptor profiling for specificity assessment.

The cyclic nucleotide phosphodiesterases (PDEs) are enzymes that catalyze hydrolysis of 3′, 5′-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5′-nucleotide monophosphates. These enzymes play important roles in controlling cellular concentrations of cyclic nucleotides and have central roles in a variety of intracellular signaling events. As such, phosphodiesterases are emerging as a promising class of drug targets, particularly in asthma, cardiovascular and CNS diseases. Currently PDE inhibitors are identified in screens employing biochemical assays using pure substrates (cAMP or cGMP) and purified recombinant PDE enzymes. We have leveraged the CNG channels in the ACTOne Technology to detect intracellular cAMP/cGMP changes and developed the first commercially available live-cell PDE inhibitor assays.

To measure cAMP specific PDE activity, a constitutively active Gs-coupled GPCR is over-expressed in the cells containing CNG channels to stimulate adenylyl cyclases, leading to cAMP synthesis. The produced cAMP is hydrolyzed by intracellular PDE so the steady cAMP is kept at low level. Upon the PDE activity inhibition, the cellular cAMP level rises quickly and is measured by the CNG biosensor. This assay has been used to identify inhibitors of PDE4 in a 1536 well format (Titus et al, J Biomol Screen 2008; 13: 609-618).

To assay cGMP specific PDE activity, soluble guanylate cyclase (sGC) was stably transfected into the cells containing CNG channels. Cellular cGMP can be increased by the sGC stimulator, BAY 41-2272. In the presence of cGMP specific PDE, the produced cGMP is hydrolyzed quickly by PDE so the steady level of cGMP is low. By adding a cGMP specific PDE inhibitor, the cGMP can accumulate quickly and activate CNG channels.

ACTOne™ cAMP Fluorimetric ELISA Assay Kit, one 96-well plate (CB-80500-503) Signal Transduction Codex Biosolutions, Inc. CDXA70003
Adenosine 3’, 5’ cyclic monophosphate (cAMP) is an important second messenger in intracellular signal transduction. Monitoring cAMP levels is one of the most common ways to screen for agonists and antagonists of GPCRs. ACTOne™ cAMP Fluorimetric ELISA Assay Kit is based on the compe..
ACTOne™ cAMP Fluorimetric ELISA Assay Kit, ten 96-well plates (CB-80500-513) Signal Transduction Codex Biosolutions, Inc. CDXA70004
Adenosine 3’, 5’ cyclic monophosphate (cAMP) is an important second messenger in intracellular signal transduction. Monitoring cAMP levels is one of the most common ways to screen for agonists and antagonists of GPCRs. ACTOne™ cAMP Fluorimetric ELISA Assay Kit is based on the compe..
ACTOne™ Membrane Potential Dye Bulk Kit (100-plate) (CB-80500-211) Signal Transduction Codex Biosolutions, Inc. CDXB40002
cAMP is a key second messenger involved extensively in cellular signal transduction pathways associated with the majority of G-protein coupled receptors (GPCRs). The activation of these GPCRs by neurotransmitters, lipids, nucleotides, peptides and hormones results in the activation or the inhibition..
ACTOne™ Membrane Potential Dye Kit (10-plate) (CB-80500-201) Signal Transduction Codex Biosolutions, Inc. CDXB40001
cAMP is a key second messenger involved extensively in cellular signal transduction pathways associated with the majority of G-protein coupled receptors (GPCRs). The activation of these GPCRs by neurotransmitters, lipids, nucleotides, peptides and hormones results in the activation or the inhibition..
Anti-Active Arf6 Mouse Monoclonal Antibody, 30 µl (26918) Signal Transduction NewEast Biosciences NEBA10223
Background ARF6 ( ADP- ribosylation factor 6 ) is a member of the ARF superfamily. ARF genes encode small GTPases that increase the ADP- ribosyltransferase activity of cholera toxin and are critical for vesicular trafficking as activators of phospholipase D. Arf6 regulate s membrane trafficki..
Anti-Active Arl1 Mouse Monoclonal Antibody, 30 µl (26924) Signal Transduction NewEast Biosciences NEBA10229
Background Arl1 (Arf-like 1) localizes to the Golgi complex, where it is believed to recruit effector proteins to the trans-Golgi network. Golgi-localized proteins golgin-97 and golgin-245 have been shown as Arl1 effectors. Additional Arl1 effectors include the GARP (Golgi-associated retrograde pro..
Anti-Active Arl3 Mouse Monoclonal Antibody, 30 µl (26925) Signal Transduction NewEast Biosciences NEBA10230
Background Arl3 (Arf-like 3) is an Arf family protein. Arl3 binds microtubules to alter specific aspects of cytokinesis. It has been proposed that retinitis pigmentosa 2 functions in concert with Arl3 to link the cell membrane and the cytoskeleton in photoreceptors as part of the cell signaling or ..
Anti-Active Cdc42 Mouse Monoclonal Antibody, 30 µl (26905) Signal Transduction NewEast Biosciences NEBA10212
Background Small GTPases are a superfamily of cellular signaling regulators. Cdc42 belongs to the Rho sub-family of GTPases that regulate cell motility, cell division, and gene transcription. GTP binding increases the activity of Cdc42, and the hydrolysis of GTP to GDP renders it inactive. GTP hydr..
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Anti-Active Gα13-GTP Mouse Monoclonal Antibody, 30 µl (26902) Signal Transduction NewEast Biosciences NEBA10209
Background Heterotrimeric G proteins are essential cellular signal transducers. Gα13 is one of the G proteins that could mediate cell migration and angiogenesis. Other biochemical and physiological functions of Gα13 are being explored. Tested Applications IP, IHC Source Species Mouse Species R..
Anti-Active Gαi Mouse Monoclonal Antibody, 30 µl (26901) Signal Transduction NewEast Biosciences NEBA10208
Background Heterotrimeric G proteins are critical cellular signal transducers. Gαi represents one sub-family of G proteins that could mediate the inhibition of adenylyl cyclases. Other biochemical and physiological functions of Gαi proteins are being explored. Tested Applications IP, IHC Sour..
Anti-Active Gαo Mouse Monoclonal Antibody, 30 µl (26907) Signal Transduction NewEast Biosciences NEBA10214
Background Heterotrimeric G proteins are critical cellular signal transducers. Gαo represents one sub-family of G proteins that could mediate the inhibition of adenylyl cyclases. Other biochemical and physiological functions of Gαo proteins are being explored. Tested Applications IP, IHC Source ..
Anti-Active Gαs Mouse Monoclonal Antibody, 30 µl (26906) Signal Transduction NewEast Biosciences NEBA10213
Background Heterotrimeric G proteins are critical cellular signal transducers. Gαs represents one sub-family of G proteins that could mediate the stimulation of adenylyl cyclases. Tested Applications IP, IHC Source Species Mouse Species Reactivity Anti-active Gαs antibody recognizes active Gα..
Anti-Active Gαz Mouse Monoclonal Antibody, 30 µl (26908) Signal Transduction NewEast Biosciences NEBA10215
Background Heterotrimeric G proteins are critical cellular signal transducers. Gαz represents one sub-family of G proteins that could mediate the inhibition of adenylyl cyclases. Other biochemical and physiological functions of Gαz proteins are being explored. Tested Applications IP, IHC Source ..
Anti-Active Rab11 Mouse Monoclonal Antibody, 30 µl (26919) Signal Transduction NewEast Biosciences NEBA10224
Background Small GTPases of the Rab- family are crucial elements of the machinery that controls membrane traffic. Rab11 has been shown to control traffic through the recycling endosome. Tested Applications IP, IHC Source Species Mouse Species Reactivity Anti-active Rab11 antibody recogniz..
Anti-Active Rab3 Mouse Monoclonal Antibody, 30 µl (26920) Signal Transduction NewEast Biosciences NEBA10225
Background Rab3 is a member of the Rab-family GTPases and is specifically targeted to synaptic vesicles. Tested Applications IP, IHC, IF Source Species Mouse Species Reactivity Anti-active Rab3 antibody recognizes active Rab3 proteins of vertebrates. Datasheet   Immunoprecipitatio..
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