Neha Khandelwal, I think your plan is correct. You need to clone the target promotor sequence of your transcription factors, upstream to the luciferase gene of the luciferase vector. Or else you can check whether they are commercially available at addgene (http://www.addgene.org/). If you plan to clone the promoter, several consecutive repeats of the promotor sequence might give promising results of the activated transcription factor. For example commercially available pNL3.2.NF-κB-RE reporter vector for NFkB activation, contains five consecutive CGGGAATTT and CCGGGGACTTTC NF-κB promotor sequences alternatively. Then you need to co-transfect both the transcription factor overexpressing vector (probably pcDNA3.1 (+)) and your target promoter-driven luciferase vector at the same time.

Dear Neha,

apperently you know the two TFs of interest and their binding motifs. Thus in your experiment you will show what is already known and Luciferase activity will go up. You will not be able to measure "TF activity" but promoter activity in the presence of those TFs. However, the idea of your experiment is not fully clear to me.

Best

Boris

@ Boris Hi. Thanks for your reply. The two transcription factors that I am working on have their promoter sequence known. I want to check the activity of endogenous TFs in cancer cells using luciferase assay. However, before I do that, I wanted to check if the reporter construct which has TF promoter is working. As when I inhibit these transcription factors by small molecules, Luciferase activity is the measure of inhibition.