Scintillation proximity assay: a versatile high-throughput screening technology

Cook, Neil D.

doi:10.1016/1359-6446(96)10026-x

Cited by 146 publications

(74 citation statements)

References 42 publications

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“…SPA beads are microspheres impregnated with a scintillant, and any radioactive product that is brought into the proximity of the bead can thus be directly monitored in a scintillation counter (7). If the radioactive substrate is not captured, then this results in an easy, homogenous assay with no separation steps.…”

Section: Resultsmentioning

confidence: 99%

Novel Scintillation Proximity Assay for Measuring Membrane-Associated Steps of Peptidoglycan Biosynthesis in Escherichia coli

Chandrakala

Elias

Mehra

et al. 2001

Antimicrob Agents Chemother

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We have developed a novel, high-throughput scintillation proximity assay to measure the membraneassociated steps (stages 2 and 3) of peptidoglycan synthesis in Escherichia coli. At least five enzymes are involved in these two stages, all of which are thought to be essential for the survival of the cell. The individual enzymes are difficult to assay since the substrates are lipidic and difficult to isolate in large quantities and analysis is done by paper chromatography. We have assayed all five enzymes in a single mixture by monitoring synthesis of cross-linked peptidoglycan, which is the final product of the pathway. E. coli membranes are incubated with the two sugar precursors, UDP-N-acetyl muramylpentapeptide and UDP-[ 3 H]-N-acetylglucosamine. The radiolabel is incorporated into peptidoglycan, which is captured using wheat germ agglutinincoated scintillation proximity assay beads. The assay monitors the activity of the translocase (MraY), the transferase (MurG), the lipid pyrophosphorylase, and the transglycosylase and transpeptidase activities of the penicillin-binding proteins. Vancomyin, tunicamycin, nisin, moenomycin, bacitracin, and penicillin inhibit the assay, and these inhibitors have been used to validate the assay. The search for new antimicrobial agents that act via the late stages of peptidoglycan biosynthesis can now be performed in high throughput in a microtiter plate.

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Section: Resultsmentioning

confidence: 99%

Novel Scintillation Proximity Assay for Measuring Membrane-Associated Steps of Peptidoglycan Biosynthesis in Escherichia coli

Chandrakala

Elias

Mehra

et al. 2001

Antimicrob Agents Chemother

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“…The model was built from homology mapping of EF-2 onto the crystal structure of Thermus thermophilus EF-G. In this model a sordarin binding site was proposed based on the position of the mutations conferring resistance to sordarin: 9 of 14 mutated residues (32,33) mapped closely on domain III, defining a possible binding pocket. Accordingly, Shastry et al (21) have recently shown in this domain a block of eight amino acids (corresponding to residues 517-524 in S. cerevisiae EF-2) that may define a sordarin specificity region in natural fungal species.…”

Section: Resultsmentioning

confidence: 99%

Identification of a Putative Sordarin Binding Site inCandida albicans Elongation Factor 2 by Photoaffinity Labeling

Domínguez

Martin

2001

Journal of Biological Chemistry

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“…The approach is based on the principles of SPA, which has been used extensively for studies on receptor/ligand-type interactions (26,27). SPA is based in part on the fact that low-energy ␤ particles have a high propensity to interact with matter and thus propagate only short distances.…”

Section: Resultsmentioning

confidence: 99%

Real-time analysis of endosomal lipid transport by live cell scintillation proximity assay

Stockinger

Castoreno

Wang

et al. 2004

Journal of Lipid Research

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Scintillation proximity assay: a versatile high-throughput screening technology

Cited by 146 publications

References 42 publications

Novel Scintillation Proximity Assay for Measuring Membrane-Associated Steps of Peptidoglycan Biosynthesis in Escherichia coli

Novel Scintillation Proximity Assay for Measuring Membrane-Associated Steps of Peptidoglycan Biosynthesis in Escherichia coli

Identification of a Putative Sordarin Binding Site inCandida albicans Elongation Factor 2 by Photoaffinity Labeling

Real-time analysis of endosomal lipid transport by live cell scintillation proximity assay

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