Luciferase, activation volume

Assay of the activities of luciferin and luciferase. Small volumes (5-50 xl) of luciferase and luciferin (Section 8.2.4) are mixed in 2 ml of 0.2 M citrate buffer, pH 6.3, or 0.2 M phosphate buffer, pH 8. The measurement is made in terms of the total light emission or the initial maximum light intensity that is reached within a few seconds. The... 

Figure 5 Quantitative analysis of tripeptide proteasome inhibitor Z-LLL on rAAV-2 transduction in mouse lung. 1x10 viral particles of AV2.RSVluciferase virus was delivered into the mouse lung by nasal inhalation. To evaluate the effects of proteasome inhibitor on gene transfer, 400 (jM Z-LLL (final concentration, representing 1% viral inoculation volume) was coadministrated at the time of viral infection. The entire lung was harvested at 8 months postinfection and luciferase activity was evaluated using a previously described protocol (12). The data represents the mean ( SEM) from four independent mice in each group.

In order to solubilize the bound luciferase, the pellet is homogenized with 20-30 volumes of cold 10 mM Tris-HCl buffer, pH 7.5, containing 1M NaCl, and the activity of the homogenate is measured. The homogenate is centrifuged and the activity of supernatant is also measured. A close agreement between the two measurements indicates that 1 M NaCl has solubilized the bound luciferase. If the two values differ significantly, a further effort of solubilization is needed (see Section C1.3). 

Data summarized in Table 21.1 demonstrate that the level of luciferase gene expression increases with an increase in the volume of injected DNA solution. The maximal level of luciferase protein achieved was approximately 350 ng/mg of extracted protein from the liver. We also demonstrated in a previous study that the volume required for an optimal transfection varies with animal weight (Liu, F. et al., 1999). The volume for a maximal level of gene expression was approximately 1.2ml, 1.6ml or 3.0ml for animals with a body weight of 11—13g, 18-20 g or 30-33 g, respectively. The estimated volume for an optimal activity in mice is approximately 8-12% of animal weight, which is higher than total blood, estimated to be 7.3% of the body weight in mice (Wu et al., 1981). 

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