Abstract
Research on the mechanisms underlying circadian rhythmicity and the response of brain and body clocks to environmental and physiological challenges requires assessing levels of circadian clock proteins. Too often, however, it is difficult to acquire antibodies that specifically and reliably label these proteins. Many of these antibodies also lack appropriate validation. The goal of this project was to generate and characterize antibodies against several circadian clock proteins. We examined mice and hamsters at peak and trough times of clock protein expression in the suprachiasmatic nucleus (SCN). In addition, we confirmed specificity by testing the antibodies on mice with targeted disruption of the relevant genes. Our results identify antibodies against PER1, PER2, BMAL1 and CLOCK that are useful for assessing circadian clock proteins in the SCN by immunocytochemistry.
Highlights
The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus generates daily rhythms in behavior, hormones and physiology
The lack of good antibodies makes it difficult to assess the impact of circadian time, phase shifting stimuli, or clock gene mutations on clock gene protein products
A previously described PER1 antibody, generated in rabbit to the amino terminus of mouse PER1, called 1177, has been used successfully in many publications [11,12,13,14,15,17,18,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35]. This antibody gives good labeling of SCN cells in mouse, and no staining in Per12/2 mice [13]
Summary
The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus generates daily rhythms in behavior, hormones and physiology. Individual cells from SCN and from many other tissues express 24-hour molecular rhythmicity that results from a transcriptional-translational feedback loop. The transcription factors, CLOCK and BMAL1, form heterodimers and bind to E-box elements in the promoters of Period (Per) 1 and Per, Cryptochrome (Cry) 1 and Cry genes. The protein products of these genes form complexes, which translocate into the nucleus and interact with the CLOCK/BMAL1 complex, resulting in repression of their transactivational activity [3,4]. Posttranslational events modify the timing of this negative feedback, providing fine control over cycle length of the molecular oscillations [5,6,7,8,9,10]
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