Identification of prothrombin from plasma as a histone-binding protein. (A) Coomassie brilliant blue staining of histone-binding proteins (black circles) captured from plasma by histone-conjugated Sepharose and separated by 2D gel. (B) Typical liquid chromatography-mass spectrometry peaks of a peptide from trypsin-digested spot from 2D gel. (C) Western blotting of isolated histone-binding proteins using antiprothrombin antibody with commercial prothrombin (ProT) as a positive control. Arrow indicates the full length of prothrombin. (D) Gel overlay assay. Equal molar concentrations (2 µmol/L) of recombinant individual human histones were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with horseradish peroxidase–conjugated prothrombin (upper panel). Lower panel: Coomassie blue–stained gel to demonstrate equal loading of proteins. (E-G) SPR curves for calculating Kd values (mean ± SD) of prothrombin with histone H2B (E), H3 (F), and H4 (G), respectively. Each experiment was repeated 3 times, and a typical experiment is presented. RU, response unit.