Accelerated nuclei preparation and methods for analysis of histone modifications in yeast.
The continuing identification of new histone post-translational modifications and ongoing discovery of their roles in nuclear processes has increased the demand for quick, efficient, and precise methods for their analysis. In the budding yeast Saccharomyces cerevisiae, a variety of methods exist for the characterization of histone modifications on a global scale. However, a wide gap in preparation time and histone purity exists between the most widely used extraction methods, which include a simple whole cell extraction (WCE) and an intensive histone extraction. In this work we evaluate various published WCE buffers for their relative effectiveness in the detection of histone modifications by Western blot analysis. We also present a precise, yet time-efficient method for the detection of subtle changes in histone modification levels. Lastly, we present a protocol for the rapid small-scale purification of nuclei that improves the performance of antibodies that do not work efficiently in WCE. These new methods are ideal for the analysis of histone modifications and could be applied to the analysis and improved detection of other nuclear proteins.
Kizer
Kelby O
KO
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
Xiao
Tiaojiang
T
Strahl
Brian D
BD
D002021:Buffers; D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D042421:Histone Code; D006657:Histones / Q000378:metabolism*; D007620:Karyometry / Q000379:methods; D009687:Nuclear Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017186:Titrimetry / Q000379:methods
D002021:Buffers; D006657:Histones; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins