luciferase concentration? - (Mar/11/2005 )
how can i determine the luciferase concentration? should i make a standard curve using Bradford assay with BSA as the standard and measure the OD of luciferase with the luminometer? after that using the standard curve to find the concentration of my luciferase samples?
any good links of luciferase assay you know? i have searched and it is not that good.
besides: how can i convert the relative light units (RLU) of luciferase into concentration? what does it mean with integration time?
I've never done a luciferase standard curve. I've done on occassions a beta-gal standard curve, but only to check that the luminometer, my reagents and my hands were working OK after a period of not doing reporter gene assays. For this beta-gal curve I've used the lyofilized beta-gal which comes on the kit I use.
Probably you won't need a standard curve. You would only need RELATIVE values of luciferase activity as usually you will be comparing two or more samples- a control one and an stimulated sample, for instance. Am I right?
What you would need is some kind of control to relate the values of luciferase in your samples to it- usually it would be beta-gal or renilla-luc expressed under the control of a constitutive strong promoter such as RSV or CMV. You should co-transfect this control with your luciferase plasmid, so that you can use the control to correct for things such as the differences in cell number or the transfection efficiency from well-to-well. You could also measure the protein concentration by Bradford instead of co-transfecting a control vector, but in that case you could not correct by transfection efficiency. Some references: Biotechniques, Biotechniques, BMC Biotechnology.
As for integration time, is the time during which you measure luciferase activity, usually 10 s. The final value you get is the sum of the light emitted during that time (the integral- the value of the area under the curve).
Hope it helps. Cheers!
should i cotransfect my plasmid and the control and then doing the dual assay or should i transfect my plasmid and the control in different samples?
how is that possible that we can correct the differences in cell number or the transfection efficiency from well-to-well by cotransfecting both plasmids in the same cell?
thanks for your excellent answers.
Hi! The idea of the control in the luciferase assay is the same as when you do semi-quantitative RT-PCR. You need to co-amplify a housekeeping gene (actin, hprt, whatever...) in the same samples in which you amplify your gene of interest, right?
In the case of the luciferase assays, let's say you're studying a promoter of interest cloned into pGL3 and you want to study the influence of different hormones on its activity, for instance. In that case you would co-transfect all your wells with the promoter-pGL3 vector + CMV-renilla vector, and then you would incubate some of your wells in serum-free media, or in serum-free media supplemented with insulin, for instance, in order to see if insulin stimulates of repress the activity of your promoter.
Thus, you will measure in each well the amount of Renilla activity (which you expect will not be changed by insulin or whatever other manipulations you're doing to the sample, only by TRANSFECTION EFFICIENCY), and the amount of Luciferase, which refelects the activity of your promoter. The value of Luciferase will change from sample to sample due to the manipulation you do (adding insulin in this case), but also because of transfection efficiency. To eliminate this unwanted efficiency of transfection efficency, for each sample you need to calculate Luciferase/Renilla (L/R). And to see the effect of insulin on your promoter, you would need to divide this L/R value from the sample treated with insulin (L/R-INS) by the same value of the untreated sample (L/R-UN):
(L/R-INS / L/R-UN)- so that you will get a RELATIVE value of activation or repression for your promoter in the presence of insulin.
I seem to have written a lot, but I don't know if I have made myself clear. Hope that it helps! Cheers!!
thank you, badcell. you explained very well.