Why is my fluorescent protein so dim?
Cells typically need only low to moderate expression of a drug resistance gene to acquire resistance to the drug. For fluorescent proteins (FPs), in contrast, high levels of expression are required to obtain bright fluorescence signal, whereas low to moderate expression may produce only weak signal or signal under detectable threshold of the hardware. In addition, certain FPs (e.g. EBFP) are much dimmer than common FPs (e.g. EGFP), and are especially difficult to detect. In fact, some researchers had to resort to immunofluorescence with anti-FP antibodies in order to visualize their FPs of interest in cell culture or in vivo. Below are some of the most frequent causes for poor fluorescence: