Reporter in this case is a Gaussia luciferase. All I know is Luciferase has a broad linear range. How does the method vary (if at all) for different cell types used for transfection?
You need to consider several relationships here to answer this question:
1) linearity of your instrument in measuring luminescence
2) relationship between enzyme and substrate concentration (i.e. is substrate maintained in vast excess or not)
3) relationship between amount of plasmid, transfection efficiency, and reporter expression
All of these can impact whether you observe a linear relationship between plasmid amount and reporter signal. In general, Gaussia is quite stable and has a very high catalytic rate. Thus you can easily end up with saturating luminescence signal in cases of a strong promoter or a high amount of expression. To check linearity of your response, simply perform serial dilutions of your Gaussia extract to see what the relationship between extract concentration and luminescence signal. If some samples are routinely falling outside the linear range, you can always correct for this by dilution.
In terms of plasmid titration, you need to be very careful because total DNA amounts affect transfection efficiency. Therefore, always hold total DNA concentration constant by using some sort of control DNA (for example, pUC19 plasmid or a promoterless Gaussia plasmid). Then you can titrate your specific reporter over a 10-fold concentration range (for example 0.2 to 2ug in a 6-well), and empirically determine the relationship between input plasmid and reporter activity.
Differences in cell lines will influence the choice of transfection reagent, total DNA, and amount of reporter utilized. Therefore, you may need to re-optimize some of these parameters if you are working in multiple cell types, particularly if one transfects much better than another, or you are using a tisse-specific promoter that shows a great deal of variability between cell types.
Membrane-free cell lysates are unpurified enzyme source and we work with the assumption that other contaminants in the lysates do not interfere with the activity of luciferase. The application therefore makes the assumption that these lysates are no different from purified enzymes. If you want to know the exact linear range then you should buy the purified enzyme and titrate it with your substrate and perform kinetic assays. The substrate will become limiting after some time if you are performing kinetic analysis with lysates rich in luciferase. The standard kits (like Promega) are linear over several order of enzyme activity. You can validate this if you are unsure. You could always use a second luciferase (there are different ones in the market now) which will serve as control for the level of heterogeneity in transfection efficiency and protein turnover. Cheers.
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