Luciferase assay

Luciferase assay. In this technique, firefly luciferase is used to measure small amounts of adenosine triphosphate (ATP) in a bacterial culture, ATP levels being reduced by the inhibitory action of aminoglycoside antibiotics. This method may find more application in the future as more active and reliable luciferase preparations become available. 

Enzymatic Assay. The enzymatic (luciferase) assay for adenosine triphosphate (ATP) is one of the methods applied to areas of biocidal control in oil production operation [1454]. A reliable method for the determination of ATP is the measurement of bioluminescence produced by the luciferin luciferase system. 

Figure 3. Influence at 500 (lM salicylic acid on ATP content of excised oat roots at four pH values. ATP determined by luciferin/ luciferase assay.

Cells are lysed for Firefly and Renilla luciferase assays using the Dual-Luciferase Reporter Assay system (Promega), following the manufacturer s instructions. We use a multimode microplate reader with automatic injectors (FLUOROstar Optima from BMG Labtech, OfFenburg, Germany) for luminescence measurements. 

Luciferase assay tubes 5-ml translucent plastic tubes for luciferase assays (75 mm x 12 mm, Sarstedt). 

Hastings J.W., Baldwin T.O., Nicoli M.Z., Bacterial luciferase assay, purification and properties, Methods Enzymol. 1978 57 135-152. 

Andre et al. [8] discuss the determination of adenosine-5 -triphosphate by luciferin-luciferase assay. This method was applied to the determination of adenosine-5 -triphosphate in bacterial colonies filtered from samples of polluted water after incubation for different periods. The adenosine-5 -triphosphate was extracted from the residue in the filter and the amount compared with the biochemical oxygen demand of the filtered water. The oxygen uptake rate and the rate of formation of adenosine-5 -triphosphate were then plotted against time, the two curves being similar in up to three to four days incubation, after which adenosine-5 -triphosphate production declined markedly, although oxygen uptake continued to increase. 

Tobin et al. [14] give details of two extraction procedures for the determination of adenosine-5 -triphosphate in sediment samples by luciferin-luciferase assay. 

Dye oxidation (e.g., tetrazolium reductase activity with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, MTT 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt, WST-1 3- (4,5 -carboxymethoxyphenyl) -2-(4-sulfophenyl)-2 H-tetra-zolium, MTS 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt, XTT 2,2 -di-p-nitrophenyl-5,5 -diphenyl-3,3 -(3,3 -dimethoxy-4,4 -diphe-nylenej-ditetrazolium chloride, NET), Alamar blue assays, ATP concentration (e.g., luciferase assay), oxygen consumption (e.g., oxygen electrodes, phosphorescent oxygen-sensitive dyes), mitochondrial protein and nucleic acid synthesis mitochondrial mass (e.g., mitotracker dyes) mitochondrial membrane potential (e.g., tetramethylrho-damine methyl ester, TMRM tetramethylrhodamine ethyl ester, TMRE)... 

Hastings Notwithstanding Michael Menaker s data on the luciferase assays on brain tissues, it is possible for various brain regions to show only one or two cycles. The accepted idea is that what is happening in the motor cortex is a reflection of activity that is being caused by the SCN. It is not an intrinsic oscillation of the motor cortex that would be sustained for any time. 

Schibler Why not just use the luciferase assay with peripheral tissue in culture ... 

Figure 21.5 Persistence of transgene expression. Animals were injected with 1,6ml saline containing 1 Opg ( ) or 50[ig (o) of either pCMV-Luc (A) or pCMVhAAT (B) plasmid DNA. For luciferase gene expression, animals were sacrificed at various time intervals and luciferase activity in the liver was determined by standard luciferase assay. For human a 1-antitrypsin gene expression, blood was collected at appropriate times and serum concentration of human q 1-antitrypsin was determined by a standard ELISA. Error bars represent s.e.m. from three mice in A and five mice in B.

For luciferase assays, use white 384-well tissue culture treated assay plates (Costar, Acton, MA). For imaging assays, use black plates with clear bottoms. Either plastic-bottomed (cat. no. 3712, Costar) or glass-bottomed (MGB 101-12, Matrical, Spokane, WA) plates can be used. 

Jones, D. P., Sherf, B. A., and Wood, K. V. (1995) Luciferase assay system vendor comparison. Promega Notes Magazine 54, 20. 

Luciferase Assay System, 100 assays, El500 (Promega, Madison, WI). 

Once at least three stable HT-29-luciferase lines are established, determine luciferase enzymatic activity in cell extracts obtained from each line using the Luciferase Assay System from Promega by following the manufacturer s instructions. Using this assay, a specific luciferase signal in the range of 107-108 luciferase counts/mg protein can be expected. 

Figure 4 Luciferase expression 48 h after transfection of HepG2 cells with an AVE/ pLuc complex in 10% serum as a function of DNA concentration. Experimental conditions were as described in Figure 3. Luciferase transgene expression was analyzed with a Pro-mega luciferase assay system, and samples were read in a Tropix TR717 microplate lumi-nometer. Cellular protein was measured using a Bio-Rad protein assay kit.

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