Use of a native affinity ligand for the detection of G proteins by capillary isoelectric focusing with laser‐induced fluorescence detection

Abstract

Affinity probe capillary isoelectric focusing (CIEF) with laser-induced fluorescence was explored for detection of Ras-like G proteins. In the assay, a fluorescent BODIPY FL GTP analogue (BGTPgammaS) and G protein were incubated resulting in formation of BGTPgammaS-G protein complex. Excess BGTPgammaS was separated from BGTPgammaS-G protein complex by CIEF using a 3-10 pH gradient and detected in whole-column imaging mode. In other cases, a single point detector was used to detect zones during the focusing step of CIEF using a 2.5-5 pH gradient. In this case, analyte peaks passed the detector in approximately 5 min at an electric field of 350 V/cm. Detection during focusing allowed for more reproducible assays at shorter times but with a sacrifice in sensitivity compared to detection during mobilization. Resolution was adequate to separate BGTPgammaS-Ras and BGTPgammaS-Rab3A complexes. Formation of specific complexes was confirmed by adding GTPgammaS to samples containing BGTPgammaS-G protein. GTPgammaS competed with BGTPgammaS for G protein binding sites resulting in decreased BGTPgammaS-G protein peak heights. The concentrating effect of CIEF enabled detection limits of 30 pM.

Application of imaging capillary IEF for characterization and quantitative analysis of recombinant protein charge heterogeneity

“…A CCD placed above the capillary column takes images of the entire capillary, which allows for real-time detection of the focusing protein inside the capillary. Several applications of recombinant monoclonal antibody [7,8] viruses [9], proteins [10] and small molecules [11] charge heterogeneity analysis using a commercial imaging cIEF instrument from Convergent Bioscience (Toronto, Canada) have been described.…”

Attomole protein analysis by CIEF with LIF detection

“…Voss and colleagues [11] extended this technology by capturing a protein against the capillary wall after focusing and then using a fluorescent antibody to tag the immobilized protein; detection limits in the low attomole range were reported for the target protein. Kennedy [12] employed a fluorescently labeled affinity reagent for characterization of selected G-coupled receptors with midattomole detection limits. Yeung [13] reported the detection of hemoglobin from single red blood cells based on native fluorescence and CIEF.…”

Capillary electrophoresis–based immunoassay and aptamer assay: A review

“…Aptamers are mostly short, single‐stranded oligonucleotides (RNA, ssDNA) with high affinity and specificity to the target molecule. Few articles were published about using of different types of molecules such as oligopeptides, monosaccharides, or copolymers as aptamers . The specificity is given by the 3D structure of aptamers, which can be characterized by stems, loops, bulges, hairpins, triplexes, or quadruplexes .…”

“…For example, G‐protein‐BODIPY FL GTPγS, hairpin‐structured DNA‐peptide nucleic acid, concanavalin A‐monosaccharides, dorzolamide‐human carbonicanhydrase II, and tubulin‐anti‐mitotic compounds pairs were examined . Other examples of the aptamer‐based ACE applications are summarized in Table .…”