Competitive adsorption of labeled and native protein in confocal laser scanning microscopy

Abstract

Confocal laser scanning microscopy has been previously applied to the study of protein uptake in porous chromatography resins. This method requires labeling the protein with a fluorescent probe. The labeled protein is then diluted with a large quantity of native protein so that the fluorescence intensity is a linear function of the labeled protein concentration. Ideally, the attachment of a fluorescent probe should not affect the affinity of the protein for the stationary phase; however, recent experimental work has shown that this assumption is difficult to satisfy. In the present study, we present a mathematical model of protein diffusion and adsorption in a single adsorbent particle. The differences in adsorption behavior of labeled and native protein are accounted for by treating the system as a two-component system (labeled and native protein) described by the steric mass action isotherm (SMA). SMA parameters are regressed from experimental linear gradient elution data for lysozyme and lysozyme-dye conjugates (for the fluorescent dyes Cy3, Cy5, Bodipy FL, and Atto635). When the regressed parameters are employed in the model, an overshoot in the labeled lysozyme concentration is predicted for Cy5- and Bodipy-labeled lysozyme, but not for Atto635-labeled lysozyme. The model predictions agree qualitatively well with recent work showing the dependence of the concentration overshoot on the identity of the attached dye and provide further evidence that the overshoot is likely caused by the change of binding characteristics due to the fluorescent label.

Confocal microscopy study of uptake kinetics of α‐lactalbumin and β‐lactoglobulin onto the cation‐exchanger SP Sepharose FF

“…Liapis et al [22] and Zhang et al [23] included an additional contribution to the transport equations from a double-layer-induced electrophoretic transport mechanism. Recently, other workers have proposed that the overshoot is caused by the displacement of weaker-binding fluorescently labelled protein by its stronger-binding unlabelled native counterpart [10,12,13,24].…”

Changes in retention behavior of fluorescently labeled proteins during ion‐exchange chromatography caused by different protein surface labeling positions

“…For example, early studies that applied CLSM to ion-exchange chromatography revealed the existence of an unexpected concentration 'overshoot' phenomenon (Dziennik et al, 2003;Hubbuch et al, 2002Hubbuch et al, , 2003, which initially was attributed to a novel non-diffusive protein transport mechanism (Dziennik et al, 2003;Liapis et al, 2001). Recent experimental and modeling studies have, however, cast doubt upon these early interpretations by revealing how protein displacement during competitive adsorption can explain the obtained intraparticle concentration profiles (Martin et al, 2005;Teske et al, 2005Teske et al, , 2006. Experimentally, bio-conjugates are diluted with a high fraction of the native protein species in order to ensure signal linearity.…”

Confocal laser scanning microscopy as an analytical tool in chromatographic research

“…2). The latter has been shown to have significant effects when considering the influence of the attached dye on the adsorption and transport behavior of the protein-dye conjugate especially for ion exchange processes [15,18,19,25,34]. In addition to the alteration of long-range electrostatic interactions, hydrophobic alterations of the surface may also play an important role but was not analyzed so far comprehensively for the majority of commercially available dyes.…”