Zinc (Zn) is an essential micronutrient for various physiological and metabolic processes in plants, although it is toxic in excess. To understand better Zn-responsive proteins, we developed a highly improved method of isoelectric focusing (IEF) in which whole lysate from Arabidopsis roots is subjected to IEF without any desalting steps. In this method, samples extracted with lysis buffer containing 1.5% SDS can also be directly applied to IEF. By applying this method to Zn-treated Arabidopsis roots, 10 up-regulated and 17 down-regulated proteins were identified, 15 of which showed a significant correlation with previously reported transcriptomic data.