By Robert A. LaRossa
A state of the art selection of leading 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 decision (titer) of particular molecules to the mobile detection of a variety of pollution, to microbiological detection and research, and the molecular organic manipulation of lux, luc, and gfp structural genes. step by step tools also are incorporated for the development of alternative recombinant organisms.
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Extra resources for Bioluminescence Methods and Protocols
3. Stability to chemicalreagents:GFP fluorescenceISretained in mild denaturants, such as 1% SDS or 8 M urea, and after fixation with glutaraldehyde, paraformaldehyde, or formalm, but fully denatured GFP is not fluorescent. GFP is very sensitive to some nail polishes used to seal cover slips (1,8); therefore, use molten agarose or rubber cement to seal cover slips on microscope slides. 0 (19) Many organic solvents can be used at moderate concentrations without abolishmg fluorescence; however, the absorption maximum may shift (20).
Temperature sensitivity of GFP chromophore formation: Mammalian cells expressing GFP have been reported to exhibit stronger fluorescence when grown at 30-33°C compared to 37’C (25,26). 7. Sensitivity: GFP, like fluorescem, has a quantum yield of about 80% (211, although the extinction coefficient for GFP is much lower. Nevertheless, m fluorescence microscopy, GFP fusion proteins have been found to give greater sensitivity and resolution than staining with fluorescently labeled antibody (8). GFP fusions have the advantages of being more resistant to photobleaching and of avoiding background caused by nonspecific binding of the primary and secondary antibodies to targets other than the antigen (8).
1 Cell extraction buffer. 5. Ahquot m volumes to be used in a single day and store frozen. 2. 125 mMKC1 Aliquot m volumes to be used m a single day and store frozen. 3. 125 mMKC1; and 5 mM glucose. Ahquot m volumes to be used in a single day and store frozen. 4. Step 3 buffer. 8; 50 mM MgS04; 5 mM EDTA. 3. Enzymes 1 Pyruvate kmase: Use a preparation substantially free of other kinase activities, especially adenylate kmase We used P 1506 type II rabbit muscle enzyme from Sigma (St. Louis, MO), which contains