Hello all,

I set up an experiment to detect TGF-Beta activity by SMAD Binding Element (SBE) reporter assay. For the reporter I used a commercial kit (BPS#60654) that contains SBE firefly luciferase vector and constitutively expressing Renilla luciferase vector. I mostly followed the manufacturer's instructions, seeding 30.000 HEK239T cells on 96-well clear bottom plate, transfection with Lipofectamine, 24h TGF-Beta treatment. But, normally, the company suggest to use their two step luciferase assay system. Instead, I used D-luciferin as a firefly substrate (note that I do not have a substrate for renilla luciferase for positive control), and Glo lysis buffer to lyse cells (the company mentions that lysis is required). So, in the experiment day, I basically removed the medium, added lysis buffer, incubated 5 min, and added the luciferin. Since the half-life of my D-luciferin is very short, I directly put it to luminometer. However, the readings are very very low. It is too low that same as empty wells (even the wells with no liquid inside at all). I also waited about 10 minutes but I still could not get any read. In case I have a problem about D-luciferin, I tried with a cell line that constitutively express firefly luciferase, and get a strong signal. My question is what would be the problem and how can I solve it.