An icosahedral virus as a fluorescent calibration standard: a method for counting protein molecules in cells by fluorescence microscopy.
The ability to replace genes coding for cellular proteins with DNA that codes for fluorescent protein-tagged versions opens the way to counting the number of molecules of each protein component of macromolecular assemblies in vivo by measuring fluorescence microscopically. Converting fluorescence to absolute numbers of molecules requires a fluorescent standard whose molecular composition is known precisely. In this report, the construction, properties and mode of using a set of fluorescence calibration standards are described. The standards are based on an icosahedral virus engineered to contain exactly 240 copies of one of seven different fluorescent proteins. Two applications of the fluorescent standards to counting molecules in the human parasite Toxoplasma gondii are described. Methods for improving the preciseness of the measurements and minimizing potential inaccuracies are emphasized.
Department of Biology, Indiana University, Bloomington, Indiana, U.S.A.
John M
JM
Murray
D000818:Animals; D002138:Calibration; D002522:Chlorocebus aethiops; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000379:methods* / Q000592:standards*; D015800:Protozoan Proteins / Q000032:analysis; D012015:Reference Standards; D012845:Sindbis Virus / Q000737:chemistry* / Q000235:genetics; D014122:Toxoplasma / Q000737:chemistry; D014709:Vero Cells
D008164:Luminescent Proteins; D015800:Protozoan Proteins
Sindbis virus; Toxoplasma gondii; counting molecules; fluorescent protein; fluorescent standard