Bioluminescence Methods and Protocols by Robert A. LaRossa

By Robert A. LaRossa

A cutting-edge choice of leading edge equipment 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 robust tools with a large choice of functions starting from the analytical choice (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 equipment also are integrated for the development of different recombinant organisms.

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4 U of guanylate kinase 2. In 13 x 100 mm test tubes mix 50 & of reaction mixture C and 200 pL of sample. 3 Incubate the tubes at 30°C for 90 mm. 4. Stop the reactions by placing the tubes m a boiling water bath for 3 min. 4. Step 2: Destruction of Other Nucleoside Triphosphafes In order to measure only the guanylates, other nucleoside triphosphates must be removed from the samples. ATP, which is added in step 1 to convert GMP to GDP, is, of course, the biggest problem. ATP is consumed in the hexokmase reaction coupled to the glucose-6-phosphate dehydrogenase reaction to ensure the complete removal of ATP: Glucose + ATP + glucose-6-phosphate + ADP Glucose-6-phosphate + NADP++ glucono-F-lactone-6-phosphate + NADPH + H+ (3) (4) UTP is also destroyed in this step by the action of uridine diphosphoglucose pyrophosphorylase: UTP + glucose- 1-phosphate --+ UDP-glucose f PP, (5) There is currently no commercially available enzyme that can be used for the removal of CTP (see Note 7).

3. 4. 50 mL of cells, using a 1-mL micropipeter. Incubate m a boiling water bath for 3 min. Transfer to an ice bath. Cool for at least 10 mm. Assay immediately, or store frozen (-2O’C) for later assay. 2. Overview of the Steps of Guanylate Determination Three separate incubations are needed to determine the amount of guanylates m a sample; one for GTP only, a second for GTP plus GDP, and a third for GTP plus GDP plus GMP. Amounts of GDP and GMP are determined by the difference m guanylate amounts in the three incubations.

In parallel ethanohc samples subjected to the same extraction procedure as the cell material), and m, the slope of the calibration curve of the internal standards (see Note 22). 4. g , Berthold) 2. , 2-l 1) fresh, as required. 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.

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