What is one challenge associated with using GFP in high-throughput screening?

Background fluorescence leading to false positives is a significant challenge when using GFP in high-throughput screening. This issue arises because the fluorescent properties of GFP can be affected by various factors in the experimental environment, including the presence of other fluorescent compounds or intrinsic fluorescence from cell components. This background fluorescence can mask the specific signal from the GFP, leading to erroneous interpretations of the results, such as identifying a compound as a potential hit when it is merely producing a false positive response.

In high-throughput screening, where numerous samples are analyzed simultaneously, it is crucial to have clear and distinct signals to accurately assess the biological activity of compounds. The presence of unwanted fluorescence complicates the detection process, making it difficult to differentiate between true positive signals and noise. Thus, managing background fluorescence is essential to enhance the reliability of screening assays utilizing GFP.