3 years ago

A simple method to measure CLOCK-BMAL1 DNA binding activity in tissue and cell extracts [version 1; referees: 2 approved, 1 approved with reservations]

Maud Gillessen, Alfred Tamayo, Pieter Bas Kwak
The proteins CLOCK and BMAL1 form a heterodimeric transcription factor essential to circadian rhythms in mammals.  Daily rhythms of CLOCK-BMAL1 DNA binding activity are known to oscillate with target gene expression in vivo. Here we present a highly sensitive assay that recapitulates native CLOCK-BMAL1 DNA binding rhythms from crude tissue extracts, which we call the Clock Protein-DNA Binding Assay (CPDBA). This method can detect less than 2-fold differences in DNA binding activity, and can deliver results in two hours or less using 10 microliters or less of crude extract, while requiring neither specialized equipment nor expensive probes. To demonstrate the sensitivity and versatility of this assay, we show that enzymatic removal of phosphate groups from proteins in tissue extracts or pharmacological inhibition of casein kinase I in cell culture increased CLOCK-BMAL1 DNA binding activity by ~1.5 to ~2 fold, as measured by the CPDBA. In addition, we show that the CPDBA can measure CLOCK-BMAL1 binding to reconstituted chromatin. The CPDBA is a sensitive, fast, efficient and versatile probe of clock function.

Publisher URL: https://f1000research.com/articles/6-1316/v1

DOI: 10.12688/f1000research.11685.1

You might also like
Discover & Discuss Important Research

Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.