Luciferase Assay - (Oct/25/2008 )

I had a question relating to luciferase assay... why does the luciferase activity have to be realted to the amount of protein present in the cell extract?

I am an undergraduate and I have never used nor been taught on how to perform transfections so I am quite confused on the protocol. In addition to my question above does anyone know of a site that can explain transfection using a luciferase assay as I am struggling to design a prototcol for it using the chemicals we have been given. We are using BHK21 cells and we have 3 days of practicals but I have no clue what we do on each day? Does transfection take more than one day?

Cheers for any input!

Yes, read "The kinetics of enzyme-catalysed reaction" in your biochem text book to get a good understanding of it.

3-4 days of work goes this way:

day -0 seed cells in multiple well culture plates to about 70% confluence by next day,

day 1 You do transfection on the next day, which include complex formation, lay the complex to cells, and change complex-containing medium after 5-6 hrs to reduce toxic effect during transfection.

day 2 you wait

day 3 After 48 hrs from transfection time to allow cells to have enough time to make luciferase from the DNA you transfered in them, you lyse the cells to make cell extract and measure the amount of light generated from the extract. From a standard curve generated using varied amount of purified luciferase you can estimate how much enzyme is in your samples. However, most time, we only interest in the ratios of the levels of the luciferase activity in different samples. So we skip the enzyme standard curve step.

Very very simply speaking, the activity of any enzyme (including luciferase) can be directely proportional to the concentration of the enzyme if in the tube there are saturating (lot of) concentrations of the substrate and any cofactors (i.e. luciferin and ATP). Of course, this is just a "rule of thumb" and lot of exceptions can be addressed. Definetively, it is worth to understand the kinetics of enzymatic reactions as previously suggested by genehunter-1

Regarding the site, at http://www.reportergene.com you will find some hard-science about luciferase and other reporters written in a very simple way.