Fluorescent dyes as protein labels in capillary electrophoresis.

摘要

Capillary electrophoresis (CE) has been utilized for the separation and quantitation of proteins. Typically, protein analysis by CE is performed using absorbance detection; however, this work utilizes laser-induced fluorescence detection (LIF) due to its numerous advantages, some of which include greater sensitivity and selectivity. Diode lasers have also been examined as an alternative to the more costly dye or gas ion lasers typically employed in LIF detection.; Since few biological molecules are natively fluorescent, they must be tagged with a fluorescent label in order to be detected via LIF detection. Common problems arising from labeling non-fluorescent species include increased sample preparation and the risk of band broadening when running the derivatized sample. As a result, this work will focus on the utilization of fluorescent dyes, some of which also exhibit excitation and emission wavelengths sufficient for use with diode lasers, that form non-covalent interactions with the analyte of interest.; Indocyanine Green (ICG) is a negatively-charged polymethine dye which has been used as a non-covalent label for globulin-type proteins, such as human serum albumin (HSA). Diode LIF detection was employed with excitation and emission at 780 nm and 820 nm, respectively. Analyses under both basic and acidic solution conditions investigated detection limits of bound and unbound dye, aggregation effects of ICG and of ICG-HSA at various concentrations, optimal buffer pH, and binding stoichiometries of dye to protein. On-column labeling experiments were also performed to minimize band broadening. Ultimately, a mixture of proteins was labeled with ICG and separated using CE diode-LIF.; Indigo Carmine (IC) is an anionic dye that binds to cationic proteins. Upon reduction of the dye, it becomes fluorescent, and so the reduced form of IC was studied as a non-covalent label with an excitation/emission couple of 488/520 nm. The proteins Cytochrome c and trypsinogen were utilized for labeling studies with IC, and conditions such as mixing order, dye stability, the use of coated columns, detection limit studies, and redox studies among others were examined.; The STP ester, BODIPY 650/665-X, which covalently binds with primary amine groups of proteins, utilizes diode-LIF for detection of the labeled species at the excitation and emission wavelengths of 650 and 665 nm, respectively. BODIPY has been studied for use in protease assays. It was used successfully to label the protein, casein, and the enzyme trypsin was introduced to monitor the extent of hydrolysis of the bound BODIPY-casein substrate.; Lastly, the use of other dyes as protein labels is discussed briefly. Fluorescein isothiocyanate (FITC) was used to label and separate a mixture of arginine and citrulline in order to monitor the extent of nitric oxide synthase activity utilizing LIF detection at 488/520 nm, excitation/emission. Allophycocyanin (APC) was also studied as a potential fluorescent conjugate for the sensitive, dual-laser detection of non-fluorescent analytes, specifically, the biotin-avidin system utilizing LIF detection at 488/520 nm, and diode-LIF detection at 650/660 nm.