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Phosphorimaging detection and quantitation for isotopic ion flux assays.

Abstract
A 96-well-microplate-based ion flux method utilizing readily available autoradiographic phosphorimaging detection is described. Nicotinic acetylcholine receptor-mediated (22)Na influx in four cultured cell lines provided satisfactory concentration-response data for epibatidine and several other nicotinic agonists. The data were consistent with data obtained using standard 6-well assays. Assays for nicotinic-receptor-mediated (86)Rb efflux produced data similar to data obtained with the (22)Na influx assay. However, assays for (45)Ca influx were not successful, although (45)Ca was readily detected and quantified. Voltage-gated sodium channel-mediated (22)Na influx in a neuroblastoma cell line allowed assay of the effects of such sodium channel activators as batrachotoxin and a pumiliotoxin B/scorpion venom combination. Phosphorimaging detection allows for reliable beta counting of up to 1,200 simultaneous samples with excellent sensitivity and is amenable for application to high-throughput screening.
AuthorsRichard W Fitch, John W Daly
JournalAnalytical biochemistry (Anal Biochem) Vol. 342 Issue 2 Pg. 260-70 (Jul 15 2005) ISSN: 0003-2697 [Print] United States
PMID15950910 (Publication Type: Journal Article)
Chemical References
  • Alkaloids
  • Batrachotoxins
  • Calcium Radioisotopes
  • Indolizines
  • Piperidines
  • Receptors, Nicotinic
  • Rubidium Radioisotopes
  • Scorpion Venoms
  • Sodium Channels
  • Sodium Radioisotopes
  • pumiliotoxin B
Topics
  • Alkaloids (pharmacology)
  • Autoradiography (instrumentation, methods)
  • Batrachotoxins (pharmacology)
  • Calcium Radioisotopes
  • Cell Line
  • Humans
  • Indolizines (pharmacology)
  • Piperidines (pharmacology)
  • Receptors, Nicotinic (drug effects, physiology)
  • Rubidium Radioisotopes
  • Scorpion Venoms (pharmacology)
  • Sensitivity and Specificity
  • Sodium Channels (physiology)
  • Sodium Radioisotopes

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