Laser‐induced fluorescence detection of non‐covalently labeled protein in capillary isoelectric focusing

Non-Covalent labeling for fluorescence detection of proteins has been investigated to increase the sensitivity of capillary isoelectric focusing using laser-induced fluorescence (LIF) detection. Non-covalently labeling fluorescent dyes, NanoOrange, Sypro red, Sypro orange, and Sypro tangerine were explored for the coupling of bovine serum albumin (BSA) and hemoglobin. Labeled proteins were studied by two complementary detection methods, viz. whole column UV and LIF detection instruments. The studies using a commercial capillary isoelectric focusing (CIEF) instrument with UV detection gave accurate pI point determination of the labeled protein, and it was confirmed that non-covalently labeled BSA focused to well characterized peaks and the related calculated pI values did not change significantly. The axial LIF detection system confirmed the formation of fluorescent labeled BSA, and an improvement of detection sensitivity of at least 10 times was achieved using LIF as compared to the UV absorption instrument.