Tyrosine-phosphorylated proteins govern a host of cell functions, such as growth, division, adhesion and motility. We previously identified a group of Nck Src homology 2 (SH2) domain-binding proteins by combining the GST-Nck1-SH2 pull-down method with two-dimensional electrophoresis (2-DE) in hepatocellular carcinoma (HCC) tissues. In the present study, different methods and conditions for key procedures of GST-Nck1-SH2 pull-down and 2-DE were investigated and optimized. High-resolution results were obtained using the following conditions: a total amount of 100 mu l GST-Nck1-SH2 fusion proteins/10 mg liver proteins to execute the pull-down procedure; 7 M urea and 2 M thiourea as lysis buffer; ultrafiltration depletion of interferential materials. Moreover, we performed a negative control experiment using GST-4T3 during the pull-down procedure, and further demonstrated that the proteins obtained using the aforementioned method interacted with Nck in a tyrosine phosphorylation-dependent manner. The optimized method offers a rapid, efficient alternative for the high-quantity screening of tyrosine-phosphorylated protein expression and solubility, which in turn facilitates future studies on tyrosine-phosphorylated proteins.