In eukaryotic cells, nucleosomes regulate DNA processes such as transcription, replication and repair. The dynamic architecture of nucleosomes is the key for understanding gene regulation. Due to...Show moreIn eukaryotic cells, nucleosomes regulate DNA processes such as transcription, replication and repair. The dynamic architecture of nucleosomes is the key for understanding gene regulation. Due to the compacted structure of nucleosome, these dynamics are a challenging to study. Fluorescence Resonance Energy Transfer (FRET) microscopy combined with a nucleosome immobilization strategy, allows to observe conformational dynamics over long time scales. However, it is not trivial to immobilize nucleosomes properly, avoiding impurities or non-targeted molecules that could perturb measuring nucleosome dynamics. Here we present an optimized sample preparation protocol for studying conformational dynamics in nucleosomes. Using an immobilization strategy based on PLL-g-PEG/PEG-biotin and NeutrAvidin, we found a limited immobilization specificity and structural integrity of nucleosomes, possibly due to the influence of the experimental conditions as the measurements settings and the surface preparation. Nevertheless, we were able to identify some nucleosome FRET dynamics using this technique. Furthermore, an innovative method for cleaning coverslips and quantifying the cleaning quality, open a window of possibilities for automated sample preparation protocols in single molecule studies.Show less
