It is important to solubilize acetone-precipitated proteins before isoelectric focusing (IEF) to achieve high resolution 2-DE gels. To resolve the maximum possible number of plant protein spots, we developed an improved solubilization buffer for plant proteins. We demonstrated that the resolution of 2-DE gels increased dramatically as the concentration of Tris-base increased, with maximum solubilization obtained at 200 mM Tris-base (Ly200T). The Ly200T buffer was more effective than the commonly used solubilization buffer containing 40 mM Tris at solubilizing acetone-precipitated plant proteins. Use of the Ly200T buffer to solubilize proteins resulted in an increase in intensity of approximately 30% of plant protein spots in the larger-than-40 kDa region of the gel. The Ly200T buffer also improved the resolution of abundant and basic proteins. Thus, the Ly200T buffer can be used to achieve greater resolution of protein spots in plant proteomics research.