Localizing transcription factors on chromatin by immunofluorescence

This article contains a detailed protocol for localizing transcription factors on Drosophila melanogaster polytene chromosomes by immunofluorescence. The large polytene chromosomes from third-instar larval salivary gland cells allow mapping of chromosome-associated proteins at high resolution. Thus, this method has been used to investigate how broadly transcription factors function and to identify and characterize cis-acting protein domains, trans-acting proteins, and trans-acting DNA elements that are necessary for chromosomal association of transcription factors. The ability to directly visualize transcription factors bound to chromosomes during a transcriptionally active stage of the cell cycle has greatly enhanced our understanding of how transcription factors function in vivo.