Luciferase media

Fig. 1.7 Spectral change of the in vitro firefly bioluminescence by pH, with Photinus pyralis luciferase in glycylglycine buffer. The normally yellow-green luminescence (Amax 560 nm) is changed into red (Xmax 615 nm) in acidic medium, accompanied by a reduction in the quantum yield. From McElroy and Seliger, 1961, with permission from Elsevier.

Fig. 1.14 Relationship between the incorporation of 180 into product CO2 and the amount of luciferin used, in the bioluminescence reaction of Cypridina luciferin catalyzed by Cypridina luciferase. The reactions were carried out in H2160 medium with 1802 gas (solid line) in H2lsO medium with 16C>2 gas (dashed line) and the control experiment of the latter using C16C>2 gas instead of luciferin and luciferase (dotted line), all in 20 mM glycylglycine buffer, pH 7.8, containing 40 mM NaCl. From Shimomura and Johnson, 1973a. Reproduced with permission from Elsevier.

The bioluminescence systems of Phengodidae (railroad worms) and Elateroidae (click beetles) are basically identical to that of Lampyridae (fireflies), requiring firefly luciferin, ATP, Mg2+ and a luciferase for light emission. However, there seem to be some differences. Viviani and Bechara (1995) reported that the spectra of the luminescence reactions measured with the luciferases of Brazilian fireflies (6 species) shift from the yellow-green range to the red range with lowering of the pH of the medium, like in the case of the Photinus pyralis luciferase (see Section 1.1.5), whereas the spectra... 

The reaction scheme of Latia bioluminescence. Based on the structures of luciferin 1 (Ln) and the product of luminescence reaction 2 (OxLn), it was proposed that the luciferase-catalyzed luminescence reaction of Latia luciferin in the presence of the purple protein results in the formation of 2 moles of formic acid, as shown in the scheme A (Shimomura and Johnson, 1968c). However, when the luminescence reaction was carried out in a medium containing ascorbate and NADH (in addition to the purple protein) to increase the quantum yield, it was found that only one mole of formic acid was produced accompanied... 

Luciferase-catalyzed luminescence of luciferin. Odontosyllis luciferin emits light in the presence of Mg2+, molecular oxygen and luciferase. The relationship between the luminescence intensity and the pH of the medium shows a broad optimum (Fig. 7.2.8). The luminescence reaction requires a divalent alkaline earth ion, of which Mg2+ is most effective (optimum concentration 30 mM). Monovalent cations such as Na+, K+, and NH have little effect, and many heavy metal ions, such as Hg2+, Cu2+, Co2+ and Zn2+, are generally inhibitory. The activity of crude preparations of luciferase progressively decreases by repeated dialysis and also by concentrating the solutions under reduced pressure. However, the decreased luciferase activity can be completely restored to the original activity by the addition of 1 mM HCN (added as KCN). The relationship between the concentration of HCN and the luciferase activity is shown in Fig. 7.2.9. Low concentrations of h and K3Fe(CN)6 also enhance luminescence, but their effects are only transient. 

Effect of pH. The light emission from most bioluminescence systems is affected by the pH of the medium, and some luciferases and photoproteins can be made inactive at certain pH ranges without resulting in permanent inactivation. For example, the luminescence of euphausiids can be quenched at pH 6, the luminescence of aequorin can be suppressed at pH 4.2-4A, and the luciferase of the decapod shrimp Oplophorus becomes inactive at about pH 4. In the case of Cypridina luminescence, however, the acidification of an extract to below pH 5 results in an irreversible inactivation of the luciferase. 

Residual molecular oxygen. The process of degassing to remove contaminating CO2 also removes most of 16C>2 in the reaction medium, and the amount of residual 16 O2 after degassing is estimated to be about 10-20 nmol in a solution of 5 ml. This residual 16C>2 can be removed by luminescence reaction when the solutions of luciferin and luciferase are mixed under evacuated conditions, or can be simply diluted with a large excess of 18C>2 to reduce its effect to a negligible level. 

The next day (48 h posttransfection), the medium is replaced with fresh DMEM containing different concentrations of compound (nM—fiM concentration range). After 12 h of incubation, the cells are washed with PBS, 40 1 of luciferase lysis buffer [100 mM KxP04 (pH 7.8), 0.2% Triton X-100] is added to each well, and the plate is incubated for 15 min at room temperature with gentle rocking. The cell extract is transferred into Eppendorf tubes and kept on ice. 

Fig. 7.3.2. Effect of estradiol and BP-5 on MCF7 cell proliferation and gene expression. MCF7 cells were grown in estrogen-depleted medium. Estradiol or BP-5 were added to cultures and cell number (proliferation), protein determination (PgR and pS2), gene expression (pS2 mRNA) and luciferase expression were estimated at the appropriate times, as indicated in the Methods Section.

The next day, infect the HT-29 cells with luciferase or mock retroviral particles. A 2.5 ml solution containing retroviral particles is either prepared fresh or obtained from a frozen aliquot (see above). Prepare polybrene as 100 x stock solution (0.8 mg/ml) in 1 x PBS (can be stored at —20°C). Add polybrene to the tubes containing the retroviral particles at a final concentration of 8 pg/ml. Aspirate the medium from the HT-29 cells in the two T25 flasks. Initiate the infection by adding 2.5 ml of the solutions containing the luciferase or mock retroviral particles with polybrene to both flasks. 

Fig. 6.5. Time-course of ATP synthesis in a suspension of PPase, ATPase liposomes. 25 /nl liposomes (about 0.07 mg protein per ml) were suspended in a medium containing 1 ml 0.2 M glycylglycine (pH 7.8), 0.2 ml luciferin/luciferase assay, 20 fjl 100 mM sodium phosphate, 10 pi 10 mM ADP and 50 pi 10 mM sodium pyrophosphate. The final concentration of MgClj was about 10 mM. At the arrow, 25 pi liposomes were added. The resulting luminescence was measured in an LKB luminometer 1250. The light output was calibrated by addition of a known amount of ATP. (From Ref. 98.)...

We reported that greater transfection efficiency in medium with serum was obtained in human cervical carcinoma HeLa cells, using (I) DC-Chol/DOPE liposomes (molar ratio, 1 2) than liposomes (1 1 or 3 2), (2) a modified ethanol injection (MEI) method to prepare liposomes than the dry-film method (13, 14), and (3) a dilution method to form lipoplex than direct mixing. The physicochemical properties of liposomes and lipoplexes can be examined by measuring particle size. Transfection efficiency was evaluated by using plasmid DNA encoding luciferase gene and the cells. 

Fig. 3. Property of gene delivery with BLs and US exposure (a) Schema of transfection mechanism by BLs and US. The mechanical effect based on the disruption of BLs by US exposure, which results in generation of some pores on plasma membrane, is associated with direct delivery of extracellular plasmid DNA into cytosol, (b) Luciferase expression in COS-7 cells transfected by BLs and US. COS-7 cells (1x10 cells/500 pLAube) were mixed wifh pCMV-Luc (5 pg) and BLs (60 pg). The cell mixture was exposed with US (Frequency 2 MHz, Duty 50%, Burst rate 2 Hz, Intensity 2.5 W/ cm. Time 10 s). The cells were washed and cultured for 2 days. Affer fhaf, luciferase acfivify was measured, (c) Effecf of US condition on transfection efficiency with BLs. COS-7 cells were exposed with US (Frequency 2 MHz, Duty 50%, Burst rate 2 Hz, Intensity 2.5 W/cm Time 0,1, 5,10 s) in the presence of pCMV-Luc (0.25 pg) and BLs (60 pg). Luciferase activity was measured as above, (d) Effect of serum on transfection efficiency of BLs. COS-7 cells in the medium containing EBS (0,10, 30, 50% (v/v)) were treated with US (Erequency 2 MHz, Duty 50%, Burst rate 2 Hz, Intensity 2.5 W/cm, Time 10 s), pCMV-Luc (0.25 pg) and BLs (60 pg) or transfected with lipoplex of pCMV-Luc (0.25 pg) and lipofectin (1.25 pg). (e) In vitro gene delivery to various types of cell using BLs and US. The method of gene delivery was same as above. S-180 mouse sarcoma cells, Colon26 mouse colon adenocarcinoma cells, B16BL6 mouse melanoma cells, Jurkat human T cell line, HUVEC human umbilical endothelial cells. Luciferase activity was measured as above. <10 RLU/mg protein, <10 RLU/mg protein Each data represents the mean S.D. n=3). L PEG-liposomes, LF Lipotectin...

Plasmid DNA solution prepare DNA solution e.g. luciferase reporter plasmid or eGFP plasmid, at a concentration of 12 pg DNA/ml by dilution of the stock solution with a serum- and supplement-free medium (e.g., RPMl 1640). 

Firefly luciferase High sensitivity. High linearity. No endogenous activity in mammalian cells. Need addition of substrate and ATP in an aerobic medium. 

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