For assays used to determine the binding affinity of H1 to nucleosomes and DNA, labeled H10 was diluted into binding buffer, consisting of 10 mM Tris pH 7.5, 150 mM KCl, 1 mM DTT, 5% Glycerol, 0.01% CHAPS, 0.01% NP-40 (nonidet p40 substitute; Fluka), to a concentration 2-fold greater than the desired final reaction concentration. The final H1 reaction concentration was 0.1 nM and 0.5 nM for nucleosomes and DNA, respectively. Nucleosomes or DNA were serially diluted in binding buffer into multiple master stocks for each order of magnitude (e.g. 0.2 nM, 2 nM, 20 nM, etc.). Each master stock was then used to create an experimental titration series 2-fold greater than the desired final concentration; typically performed in 200 µl PCR tubes (Genemate) or 384-deepwell low binding polypropylene plates (Eppendorf). For each replicate titration series, 20 µl of every 2-fold substrate dilution was then pipetted into a single row of the microplate using a 12-channel pipettor followed by 20 µl of the 2× H1 stock. The microplate was then quick spun at 500–2000 rpm, shaken for 2 min (VortexGenie 2) at speed no higher than 2, allowed to incubate 15–20 min at room temperature, and then imaged on a Typhoon Trio multimode imager (GE Healthcare). Images were collected with a 488 nm excitation laser and a 520 bp40 emission filter at 600 V PMT and scanned at +3 mm with press ‘on’ at 100 µm. Images were analyzed with the Array Analysis function of ImageQuant TL software by making an array with squares slightly smaller than the dimensions of each well. The data from each well were plotted in GraphPad Prism software and fit to a single exponential binding curve with a Hill coefficient using the following equation, based upon Reaction Scheme 1, where P is protein and L is the ligand: