Luciferase Assay: Scraping Cells - Equipment, Damage?

[sobored]

my protocol says;

"harvest the cells by scraping for luciferase assay"

which equipment should i use to scrap my cells? won't my cells get damaged this way?

thanks.

 

[Monique]

There are special plastic scrapers used in tissue culture, if you scrape carefully the cells won't damage too much. And your cells need to by lysed anyway before a luciferase assay, so depending whether you are going to do wash steps after scraping it won't matter that the cells damage.

 

[sobored]

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.

thanks.

 

[Monique]

I have used dual luciferase assay from Promega before, you can find the protocol here http://www.promega.com/tbs/tm040/tm040.html . It works really well. For tranfection I used FuGene reagent, for difficult cell types I used electroporation by Amaxa.

You measure luciferase expression with a luminometer, relative to the expression of a thymidine kinase promotor (which has a robust expression in a wide variety of cell types), thus the name 'dual luciferase'.

 

[sobored]

how can i convert the relative light units (RLU) of luciferase into concentration? what does it mean with integration time?