By Robert A. LaRossa
A state of the art choice of cutting edge tools for using luciferase and eco-friendly fluorescent protein in analytical biochemistry, molecular toxicology, environmental tracking, and cell-based assays. The contributors-all hands-on researchers who've perfected their protocols to a excessive measure of reproducibility-illustrate those strong equipment with a large choice of purposes starting from the analytical selection (titer) of particular molecules to the mobile detection of a variety of toxins, to microbiological detection and research, and the molecular organic manipulation of lux, luc, and gfp structural genes. step by step tools also are integrated for the development of alternative recombinant organisms.
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Additional info for Bioluminescence Methods and Protocols
1987) An analysis of 5’-noncoding sequences from 699 vertebrate messenger RNAs Nuclezc Aczds Res. 15, 8 125-8 148. 17. Niswender, K. , Blackman, S. , Magnuson, M. , and Piston, D. W. (1995) Quantitative imaging of green fluorescent protein in cultured cells, comparison of microscopic techniques, use m fusion proteins and detection limits. J. Mcrosc. 180(2), 109-l 16. 18. Inouye, S. and Tsuji, F I. (1994) Evidence for redox forms of the Aequorea green fluorescent protein FEBS Lett 351,211-214. 19.
Note that the difference between duplicates within a particular experrment was very low < 10%) In the presence of a biological matrix, the sensitivity is likely to be lower than with aqueous standards (which makes the use of internal standards necessary). Thus, in the case of measurements m extracts from cardiomyocytes (which represent a complex, protein-rich matrix), the sensitivity limit was -70 pmol malate and -90 pmol glucose-6-phosphate (and allowed the accurate measurement of cellular metabolite concentrations corresponding to values found by others in whole hearts; see refs.
Although some of them may be stable for hours to days (at least when kept at 4”C), we find it preferable for them to be freshly made to avoid, for instance, bacterial contammatlon 3. Citrate synthase, as well as malate dehydrogenase (from Boehrmger) are provided as suspensions in amrnomum sulfate. This salt was (partly) removed by spinning down the required amount of enzyme suspension and dissolving the pellet in phosphate buffer. However, prehmmary experiments have shown that the luminometrlc measurement of malate is apparently not affected by small amounts of ammonium sulfate (up to 20 mM).
Bioluminescence Methods and Protocols by Robert A. LaRossa