Study of protein-protein binding reaction by whole-column fluorescence-imaged CIEF

Whole-column fluorescence-imaged CIEF was applied to study protein-protein binding reaction. A homemade whole-column fluorescence-imaged CIEF experimental setup was built, and its CIEF performance was evaluated with native fluorescent protein green fluorescence protein and fluorescently labeled proteins (bovine albumin, human albumin, and BSA). pIs, focusing time, detection limits, and linear quantitative range of the proteins were obtained. Furthermore, the method was used to study FITC-protein A-human IgG binding reaction. Experimental results showed that the apparent binding ratio of the FITC-protein A to human IgG was 1:2, and pI of the binding conjugates were about 6.3-6.5. No binding reaction was found between green fluorescence protein and the fluorescent-labeled proteins.