Automated Kinetic Exclusion Assays to Quantify Protein Binding Interactions in Homogeneous Solution

Blake, Robert C.; Павлов, А. В.; Blake, Diane A.

doi:10.1006/abio.1999.4176

Cited by 111 publications

(90 citation statements)

References 33 publications

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“…The supported bilayers were incubated in a 1-10 g͞ml solution of antibiotin antibodies for at least 20 min, sufficient to approach saturation of biotin binding sites (23). Unbound antibodies were washed away, leaving a monolayer of protein bound to the supported bilayer (Fig.…”

Section: Methodsmentioning

confidence: 99%

Protein patterns at lipid bilayer junctions

Parthasarathy

Groves

2004

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

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We introduce a simple intermembrane junction system in which to explore pattern and structure formation by membrane-bound proteins. The junction consists of a planar lipid bilayer to which one species of protein (an IgG antibody) is bound, forming a 2D, compressible fluid. Upon the adhesion of a second lipid bilayer, the formerly uniformly distributed proteins rapidly reorganize into patterns of dense and sparse zones. Using a combination of complementary imaging techniques (fluorescence microscopy, fluorescence interference contrast microscopy, and fluorescence resonance energy transfer), we reconstruct the 3D structure of these intermembrane patterns with nanometer-scale topographic resolution, revealing the orientation of the proteins. The patterns form as the rapid bilayer-bilayer adhesion, often radiating outward from an initial, circular contact site, pushes aside the antibodies, sweeping them into areas of high density and clearing low-density regions. Coarsening of these local features is energetically costly and therefore kinetically trapped; the patterns do not change over tens of minutes. These studies demonstrate that membrane mechanical forces alone, i.e., in the absence of specific biochemical interactions, can drive m-scale organization of membrane proteins.

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Section: Methodsmentioning

confidence: 99%

Protein patterns at lipid bilayer junctions

Parthasarathy

Groves

2004

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

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“…For each of the LPA species, samples containing either 0.5 or 10 nM LT3015 IgG and 13 nM or 10 M FAF-BSA (dependent on experiment) in direct detection of the ligand-free binding sites in a sample. This technique is compatible with a variety of biological systems and has several advantages over other methods (30)(31)(32)(33) (36), to investigate S1P binding apoM in the context of isolated human HDL and LDL particles. Sevvana et al (37) showed that S1P quenched the intrinsic fluorescence of purified recombinant human apoM with an IC 50 value of 0.9 M.…”

Section: Serum Albumin Affinity Experimentsmentioning

confidence: 99%

A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay

Fleming

Glass

Lackie

et al. 2016

Journal of Lipid Research

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(GPCRs) (1-3). Many physiological processes, such as cell growth, differentiation, survival, motility, and angiogenesis (3), and pathophysiological processes, such as cancer, cardiovascular disease, multiple sclerosis, neuropathic pain, and fibrosis (4, 5), involve S1P or LPA signaling. The S1P and LPA pathways are validated therapeutic targets; many drugs and pharmacological agents have been developed to modulate the activity of receptors and enzymes in these pathways (1,4,6). Many of these compounds block circulating S1P and LPA from binding and activating cognate membrane-bound receptors.Circulating S1P exists primarily bound to carrier molecules, including HDL, LDL, and serum albumin. HDL is a protein-rich lipoprotein containing multiple protein constituents (7) and reportedly binds 50-70% of plasma S1P, whereas serum albumin reportedly binds 30% or more (8-10) . apoM represents the main protein component in HDL responsible for binding S1P, and the X-ray cocrystal structure of recombinant human apoM in complex with S1P has been solved (11). Human plasma contains approximately 0.9 M apoM (11, 12), where >95% of the total apoM occupies 5% of the HDL (apoM-HDL) and <2% of the LDL (apoM-LDL) in plasma (13,14); this stoichiometry results in less than 1 mol of S1P per mole of HDL in human plasma (15). S1P-associated HDL stimulates cellular Abstract Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive signaling lysophospholipids that activate specific G protein-coupled receptors on the cell surface triggering numerous biological events. In circulation, S1P and LPA associate with specific carrier proteins or chaperones; serum albumin binds both S1P and LPA while HDL shuttles S1P via interactions with apoM. We used a series of kinetic exclusion assays in which monoclonal anti-S1P and anti-LPA antibodies competed with carrier protein for the lysophospholipid to measure the equilibrium dissociation constants (K d ) for these carrier proteins binding S1P and the major LPA species. Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive lysophospholipids that bind and signal through multiple G protein-coupled receptors

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“…The two instruments described herein and shown in Figure 1 have the ability to meet many of the analytical needs of field investigators: they are relatively portable, easy to operate, acceptably precise, and amenable to the analysis of a wide range of contaminants. Both instruments are flow fluorimeters that use the previously described kinetic exclusion principle [1][2][3]. This method is a variation of a competitive immunoassay and the uranium assay developed for these instruments utilizes an antibody that binds to chelated U(VI) [4].…”

Section: Introductionmentioning

confidence: 99%

Field-Based Detection and Monitoring of Uranium in Contaminated Groundwater using Two Immunosensors

Melton

Williams

et al. 2009

Environ. Sci. Technol.

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Field-based monitoring of environmental contaminants has long been a need for environmental scientists. Described herein are two kinetic-exclusion based immunosensors, a field-portable sensor (FPS) and an Inline senor, that were deployed at the U.S. Department of Energy, Integrated Field Research Challenge Site in Rifle, CO. Both sensors utilized a monoclonal antibody that binds to a U(VI)-dicarboxyphenanthroline complex (DCP) in a kinetic exclusion immunoassay format; these sensors were able to monitor changes of uranium in groundwater samples from ~ 1µM to below the regulated drinking water limit of 126nM (30ppb). The FPS is a battery-operated sensor platform that could determine the uranium level in a single sample in

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Automated Kinetic Exclusion Assays to Quantify Protein Binding Interactions in Homogeneous Solution

Cited by 111 publications

References 33 publications

Protein patterns at lipid bilayer junctions

Protein patterns at lipid bilayer junctions

A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay

Field-Based Detection and Monitoring of Uranium in Contaminated Groundwater using Two Immunosensors

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