Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence.

Ullman, Edwin F.; Kirakossian, Hrair; Singh, Sharat; Wu, Zai‐Sheng; Irvin, Benjamin R.; Pease, John; Switchenko, Arthur C.; Irvine, Jennifer D.; Dafforn, Alan; Skold, C N

doi:10.1073/pnas.91.12.5426

Cited by 305 publications

(236 citation statements)

References 14 publications

(17 reference statements)

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“…To verify whether G1R and either NF-B1 p105 or SKP1A can interact in mammalian cells, their association was further analyzed in transfected cells, using full-length constructs and the AlphaScreen technology (PerkinElmer) (25,26). The AlphaScreen assay system utilizes 2 different bead types, described as donor beads and acceptor beads.…”

Section: Resultsmentioning

confidence: 99%

“…The amplified luminescent proximity homogeneous assay (AlphaScreen; PerkinElmer) (25,26) is a bead-based technology for screening biomolecular interactions in a microplate format (50). The assay utilizes 2 different bead types, described as donor beads and acceptor beads, and the surfaces of these beads are coated with reactive aldehyde groups to which biomolecules can be covalently conjugated.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Proteomic screening of variola virus reveals a unique NF-κB inhibitor that is highly conserved among pathogenic orthopoxviruses

Mohamed

Rahman

Lanchbury

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Identification of the binary interactions between viral and host proteins has become a valuable tool for investigating viral tropism and pathogenesis. Here, we present the first systematic protein interaction screening of the unique variola virus proteome by using yeast 2-hybrid screening against a variety of human cDNA libraries. Several protein-protein interactions were identified, including an interaction between variola G1R, an ankryin/F-box containing protein, and human nuclear factor kappa-B1 (NF-B1)/p105. This represents the first direct interaction between a pathogen-encoded protein and NF-B1/p105. Orthologs of G1R are present in a variety of pathogenic orthopoxviruses, but not in vaccinia virus, and expression of any one of these viral proteins blocks NF-B signaling in human cells. Thus, proteomic screening of variola virus has the potential to uncover modulators of the human innate antiviral responses.ankyrin repeats ͉ Skp1 ͉ yeast 2-hybrid

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Proteomic screening of variola virus reveals a unique NF-κB inhibitor that is highly conserved among pathogenic orthopoxviruses

Mohamed

Rahman

Lanchbury

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…DgkB Vesicle Binding Assay-The AlphaScreen assay is a bead-based technology developed for assessing biomolecular protein-target interactions in a homogeneous microplate format (21,22). AlphaScreen binding assays were developed to investigate protein-protein interactions (23), and we have developed an assay for DgkB-phospholipid interactions based on the same principles.…”

Section: Methodsmentioning

confidence: 99%

Molecular Determinants for Interfacial Binding and Conformational Change in a Soluble Diacylglycerol Kinase

Jerga

Miller

White

et al. 2009

Journal of Biological Chemistry

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DgkB is a soluble diacylglycerol (DAG) kinase that is essential for membrane lipid homeostasis in many Gram-positive pathogens. Anionic phospholipids, like phosphatidylglycerol (PtdGro), were required for DgkB to recognize diacylglycerol embedded in a phospholipid bilayer. An activity-independent vesicle binding assay was used to determine the role of specific residues in DgkB-PtdGro interactions. Lys 15 and Lys 165 were required for DgkB to dock with PtdGro vesicles and flank the entrance to the DgkB active site. Mg 2؉ was required for vesicle binding. The compromised vesicle binding by mutants in the key asparate residues forming the structural Mg 2؉ -aspartatewater network within the substrate binding domain revealed that interfacial binding of DgkB required a Mg 2؉ -dependent conformational change. DgkB interaction with phospholipid vesicles was not influenced by the presence of ATP, but anionic vesicles decreased the K m of the enzyme for ATP. Arg 100 and Lys 15 are two surface residues in the ATP binding domain that were necessary for high affinity ATP binding. The key residues responsible for the structural Mg 2؉ binding site, the conformational changes that increase ATP affinity, and interfacial recognition of anionic phospholipids were identical in DgkB and the mammalian diacylglycerol kinase catalytic cores. This sequence conservation suggests that the mammalian enzymes also require a structural divalent cation and surface positively charged residues to bind phospholipid bilayers and trigger conformational changes that accelerate catalysis.The diacylglycerol (DAG) 2 kinase superfamily (Pfam00781) defines a group of soluble lipid kinases that phosphorylate membrane-associated DAG and function in regulating intracellular phospholipid metabolism and signaling. The mammalian family members share a conserved catalytic core domain that is associated with a variety of modules that confer intracellular membrane targeting specificity (for reviews, see Refs. 1 and 2). Staphylococcus aureus DgkB is a prototypical member of Pfam00781 that has the catalytic core of the DAG kinase superfamily, but lacks the ancillary domains characteristic of its mammalian cousins. DgkB proteins are essential in Gram-positive bacteria to re-introduce the large amount of DAG arising from lipoteichoic acid biosynthesis into the phospholipid biosynthetic pathway (3). DAG is exclusively found in the membrane phospholipid bilayer and the soluble DgkB must be capable of interfacial catalysis despite the absence of the membrane targeting modules present in its mammalian counterparts.Interfacial activation is a key feature of soluble proteins that act on substrates embedded in a phospholipid bilayer and has been extensively studied in phospholipid hydrolases (4, 5). These enzymes can utilize monomeric substrates, but their activity increases significantly when substrate is presented in micelles or unilamellar vesicles. Electrostatic effects are crucial to interfacial recognition by the soluble phospholipase A 2 proteins (4), and the structu...

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“…Carbohydrate solution assay is a highthroughput, homogenous and sensitive method to characterize protein-carbohydrate interactions and glycostructures by in-solution proximity binding with photosensitizers (Figure 1). The technology, also called AlphaScreen TM , is first described by Ullman et al, and has been used to study interactions between biomolecules [30][31][32][33][34]. In these assays, a light signal is generated when a donor bead and an acceptor bead are brought into proximity.…”

Section: Carbohydrate Microarraymentioning

confidence: 99%

Carbohydrate Microarray

Chang¹

2012

Carbohydrates - Comprehensive Studies on Glycobiology and Glycotechnology

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Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence.

Cited by 305 publications

References 14 publications

Proteomic screening of variola virus reveals a unique NF-κB inhibitor that is highly conserved among pathogenic orthopoxviruses

Proteomic screening of variola virus reveals a unique NF-κB inhibitor that is highly conserved among pathogenic orthopoxviruses

Molecular Determinants for Interfacial Binding and Conformational Change in a Soluble Diacylglycerol Kinase

Carbohydrate Microarray

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