Purification and Analysis of PERIOD Protein Complexes of the Mammalian Circadian Clock

Abstract In mammals, circadian rhythms are generated at least in part by a cell-autonomous transcriptional feedback loop in which the three PERIOD (PER) and two CRYPTOCHROME (CRY) proteins inhibit the activity of the dimeric transcription factor CLOCK-BMAL1, thereby repressing their own expression. Upon nuclear entry, the PER and CRY proteins form a large protein complex (PER complex) that carries out circadian negative feedback by means of at least two basic functions: (1) it brings together multiple effector proteins that repress transcription and (2) it delivers these repressive effectors directly to CLOCK-BMAL1 bound to E-box sequences of circadian target genes. At present, the composition, mechanisms of action, and dynamics of PER complexes in circadian clock negative feedback are incompletely understood. Here, we describe several experimental approaches to the study of PER complexes obtained from mammalian tissues. We focus on the isolation of nuclei from mouse tissues, the extraction of PER complexes from the isolated nuclei, characterization of native PER complexes by gel filtration and blue native polyacrylamide gel electrophoresis, preparative immunoaffinity purification of PER complexes for mass spectrometric identification of constituent proteins, and chromatin immunoprecipitation to monitor the recruitment of PER complex proteins to CLOCK-BMAL1 at E-box sites of clock-regulated genes.