Quick and Facile Preparation of Histone Proteins from the Green Microalga Chlamydomonas reinhardtii and Other Photosynthetic Organisms

Publication date: Available online 31 January 2020Source: MethodsAuthor(s): Amanda L. Wong, Nicholas N. Totah, Anthony T. Iavarone, James J. PesaventoAbstractThe development of universal, broadly applicable methods for histone extraction from animal cells and tissues has unlocked the ability to compare these epigenetic-influencing proteins across tissue types, healthy and diseased states, and cancerous versus normal cells. However, for plants and algae, a quick and easily implemented histone extraction method has yet to be developed. In this paper, we report an optimized method that provides a unified approach to extract histones for the green microalgal species Chlamydomonas reinhardtii and Scenedesmus dimorphus as well as for corn leaf tissue. Histone extraction methods include treatment with high salt concentrations and acidification. Preparations of nuclei and whole histone proteins can be completed in about 7 hours (about 3.5 hours each) and can be performed on separate occasions. To examine the efficiency of the methods provided, we performed both qualitative and quantitative analysis of salt and acid-extracted whole histone proteins (SAEWH) via SDS-PAGE gel electrophoresis and intact protein mass spectrometry. SDS-PAGE analysis indicated that histone yields decrease when cell walls remain intact relative to cell-wall-less mutants. Using top-down mass spectrometry (TDMS) for intact protein analysis, we confirmed the presence of H4K79me1 in multiple algal species; howeve...
Source: Methods - Category: Molecular Biology Source Type: research