GFP and luciferase measurements - (Sep/21/2011 )

hey everybody,

I have a technical question. I transfected my cells with p21-luc plasmid and a GFP plasmid as a control for transfection efficiency. To determine the transfection efficiency I found in literature that I should divide the luciferase activity with the average GFP intensity.
Now, to obtain both these values I need to measure both luciferase activity (with a promega assay) and the GFP intensity (with FACS).
I am working in 24 well plates (each condition in triplicate wells) and when I want to measure GFP with facs then I need to trypsinize the cells, collect them in a tube, centrifuge, and resuspend in PBS. However for luciferase assays you need to lyse your cells and the easiest way is directly on your plate.

I was wondering if anyone has any suggestions on how to combine these two protocols. I was thinking of maybe trypsinizing my cells and dividing each sample/condition (=3wells) over two tubes, one for GFP-assay one for luciferase-assay. But then I don't really know how I should lyse my cells when they are in this tube. Is this possible? and if yes, does anyone have an idea of volumes for the lysis buffer and reagent when you start from a collection of cells in a tube? Or am I completely on the wrong path and is there a better way to do this?

It would be nice to hear any suggestions

kind regards,

Annelien

Hi Annelien,

the simplest way, if possible, is to change the normalization plasmid with a Renilla luciferase one.
by doing so, you could measure in the same lysate the two luciferase activities (Firefly P21 first, with luciferin) and Renilla then (with coelenterazime).

Dual luciferase assay kits exist as well.

Hello Annelien,
I undetand the utility of the GFP co-trasnfection control to see that trasnfeciton ws successful, but I would not recommned using it as a control reporter for normalization. GFP intensity is not a great quantitative measurement and individual cells will vary greatly in their intensity. For this reason, a Dual-Luciferase Assay would be an easier process and hopefully give more useable data. By using two luciferase, one protocol is used to lyse the cells and measure the luciferases from the single sample. The article linked here might be of some interest;
http://www.promega.com/resources/articles/pubhub/cellnotes/normalizing-genetic-reporter-assays/

Hope this is useful. Please feel free to contact me or Promega Technical Services if you have any questions on this or other procedures. We are always available to help.

Kevin

Hi everyone,

I'm doing a Renilla luciferase assay to measure the activity of protein promoter "A", and I'm transfecting with another plasmid, which is the protein "A" fused to GFP, so my question is, if I have some measures problems when measuring luciferase activity with luminometer, I mean GFP could interfere with the measurement of luciferase?

Please help me