Medline

Fluorescence from the beads was easily detectable by fluorescence photomicroscopy when the amount of translation reaction added to an immunoprecipitation corresponded to as little as 1 nl per bead (Fig. 1c). This is equivalent to about 2 × 10 6 protein molecules per bead, giving a surface density of about 80 molecules per ␮m 2. Even a density as low as about 8 molecules per ␮m 2 could be seen in the microscope (data not shown). The true density of fluorescent molecules may be even lower than indicated, as the fraction of synthesized molecules that adopted a fluorescent conformation is not known. Each bead is somewhat larger than a eukaryotic cell (90 ␮m in diameter), suggesting that a similarly small number of GFP-fusion molecules could be detected in a living cell, particularly if the protein were localized; autofluorescence from a cell, however, might place a lower limit on this number [3]. Cell-free expression of GFP seems ideal for the rapid development of new GFP mutants with brighter fluorescence or shifted spectra, based on the recently determined crystal structure [4,5]. Large numbers of mutants could be screened rapidly by using cell-free coupled transcription–translation of cDNA clones in microtiter plates. In addition, the fusion of GFP to other proteins provides a simple nonradioactive method of measuring translation efficiency, which could expedite the optimization of translation of any protein of interest. Furthermore, if radiolabeled amino acids are incorporated, fluorescence intensity can be correlated with protein quantity, allowing the determination of the density of GFP-fusion protein molecules in a microscopic image of a living cell, a task which cannot easily be accomplished by analysis in vivo. imaging of green fluorescent protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits. messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Gazetteer Medline What is it famous for? In North America, at least, it is the usual source of bibliographic information for biomedical scientists. They expect the titles and abstracts of papers in any worthy journal to be included in Medline in a timely fashion. What should it be infamous for? The fact that there are still large gaps in its coverage of the 1996 literature, but that few people realize this. The problem arose because of a labour dispute a year ago, which stopped data entry for so long that the backlog has still not been cleared. …