I was working with luciferase assay to see the ability of my compound as a ligand for receptor. The result somehow does not follow dose dependent manner. it reachs optimum fold change at low concentration and decrease after that ( in this case the optimum concentration is 1.56 microg/ml). After 1.56 microg/ml, as the concentration increase, the luciferase activity decrease. I have come across regarding the high affinity ligand binding. Does anyone have other opinions? I need more explanation of why the luciferase activity decrease. Tq in advance. I would appreciate if there is an attachment of journal too. Tq
Dear Zuriah,
The phenomenon that you observed in your experiment is well documented. Please read the publication contained in the following link with special attention to Figure 1:
https://malariajournal.biomedcentral.com/articles/10.1186/1475-2875-11-369
Hoping this will be helpful,
Rafik
Dear Zuriah Ismail,
Some information seem to be missing in your query.
1. Have you checked if your ligand exerts cytotoxicity in the cells? Because if the ligand is highly toxic for the cells, you may not get optimum number of cells to produce significant luminescence and you will getting erroneous data. Also you can optimize the transfecting agent because that is another factor which is toxic to cells. You can subculture the cell in a larger amount and repeat the experiment.
2. You should always check the transfection efficiency because certain cell lines are very hard to transfect. Do the luciferase assay at higher transfection efficiency say 80-90%.
3. You can also transfect a constitutive vector as internal control and see if that remains same for the ligand concentration.
Best of luck.
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