Schematic of in situ purification of His-tagged proteins on protein microarray slides. A) Flow diagram from recombinant protein expression to in situ purification and protein microarray. Cells were cultured in 1 mL 2xYT media in a 96-well block and the target proteins were overexpressed by adding IPTG at O.D.600 nm = 0.7–0.9. After induction, the cells were divided into several 96-well plates, and the cell pellets were stored at -20°C. The cell pellets were resuspended, and lysed. Soluble fractions of lysates were subjected to in situ purification. Metal ions, either Ni2+ or Cu2+, were attached to the surface of a chip with a chelate structure. The chip surface was blocked with PBS buffer containing 5 mM imidazole and 3% dry milk, and the E. coli endogenous proteins were washed off with PBST, pH 7.4. The chips can be either stored or applied for protein microarray application directly. B) Scheme of selective immobilization of recombinant proteins. Approximately 50 nL of each soluble fraction of cell lysates was spotted on the metal ion (Ni2+ or Cu2+) chelated chip. His-tagged recombinant proteins were immobilized by binding tightly to the metal chelate. The endogenous E. coli proteins were washed with PBS containing 0.05% Tween 20.