Sensitive\nDetection of Histones and γ‑H2AX\nby Immunoblotting: Problems and Solutions

Histones and their posttranslational modifications (PTMs)\nare critical\nregulators of gene expression. Differentiation, environmental stressors,\nxenobiotics, and major human diseases cause significant changes in\nhistone variants and PTMs. Western blotting is the mainstay methodology\nfor detection of histones and their PTMs in the majority of studies.\nSurprisingly, despite their high abundance in cells, immunoblotting\nof histones typically involves loading of large protein amounts that\nare normally used for detection of sparse cellular proteins. We systematically\nexamined technical factors in the Western-blotting-based detection\nof human histones with >30 antibodies. We found that under multiple\nprotein transfer conditions, many histone epitopes on polyvinylidene\nfluoride (PVDF) membranes had a very low antibody accessibility, which\nwas dramatically increased by the addition of a simple denaturation\nstep. Denaturation of membrane-bound proteins also enhanced the specificity\nof some histone antibodies. In comparison to standard PVDF membranes,\nthe sensitivity of histone detection on standard nitrocellulose membranes\nwas typically much higher, which was further increased by the inclusion\nof the same denaturation step. Optimized protocols increased by >100-times\ndetection sensitivity for the genotoxic marker γ-H2AX with two\nmonoclonal antibodies. The impact of denaturation and nitrocellulose\nuse varied for different histones, but for each histone, it was generally\nsimilar for antibodies targeting N-terminal and C-terminal regions.\nIn summary, denaturation of membrane-bound histones strongly improves\ntheir detection by Westerns, resulting in more accurate measurements\nand permitting analyses with small biological samples.