Protein-Labeling Effects in Confocal Laser Scanning Microscopy

Abstract

Confocal laser scanning microscopy (CLSM) is being increasingly used for observing protein uptake in porous chromatography resins. Recent CLSM studies have revealed the possible existence of a nondiffusive protein transport mechanism. Observing protein uptake with CLSM requires labeling the protein with a fluorescent probe. This study examines the effect of the probe identity on the subsequent CLSM adsorption profiles. The adsorption of lysozyme conjugated with different fluorescent probes (Cy5, BODIPY FL, Atto 635, and Atto 520) on SP Sepharose Fast Flow was measured using CLSM and zonal chromatography experiments. Results from zonal chromatography show that the retention time of lysozyme-dye conjugates differ significantly from unlabeled lysozyme. The change in retention of lysozyme upon conjugation with a fluorescent probe is consistent with the difference in net charge between the lysozyme-dye conjugate and unlabeled lysozyme. The adsorption profiles measured by CLSM show significantly different behavior depending upon whether the lysozyme-dye conjugate is retained longer or shorter than the unlabeled lysozyme. These results strongly suggest that the lysozyme concentration overshoot observed in previous CLSM experiments is the result of displacement of weaker binding labeled lysozyme by stronger binding unlabeled lysozyme.

Chromatography of proteins on charge-variant ion exchangers and implications for optimizing protein uptake rates

“…Confocal microscopy results for a basic protein (lysozyme) labeled with acidic fluorophores clearly show diffuse fronts at higher ionic strengths despite the presence of labeled and unlabeled proteins as well as relatively sharp isotherms [19]. Therefore, the presence of a diffuse front in this system appears to be a reliable indicator of a homogeneous diffusion mechanism, and the results of Teske et al [57] suggest that this will be the case whenever the labeled protein is less strongly retained than the unlabeled one. Additional support for inferring a change in uptake mechanism that reflects an increase in the uptake rate is provided by uptake measurements on pure unlabeled protein in batch and breakthrough experiments; some additional data of this kind are presented below.…”

“…Initial arguments invoked electrokinetic phenomena in various forms [19,54], but more recent theoretical and experimental studies in various groups have implicated displacement mechanisms involving labeled and unlabeled proteins [55][56][57]. Specifically, conjugation of a label such as Cy5 to a protein via a lysine residue replaces a positive charge at neutral pH by a negative charge, making the charge on the labeled protein different to that on the unlabeled one by -2.…”

“…Specifically, conjugation of a label such as Cy5 to a protein via a lysine residue replaces a positive charge at neutral pH by a negative charge, making the charge on the labeled protein different to that on the unlabeled one by -2. As a result, for a positively charged protein in cation exchange, the labeled form adsorbs more weakly than the unlabeled one, and indeed the two forms can be separated under suitably chosen conditions for ion-exchange [56,57]. During uptake, the labeled protein can be displaced by the unlabeled one, which is present in large excess, giving rise to the overshoot behavior in the presence of an otherwise sharp front [55,56].…”

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.…”

Protein adsorption kinetics in charged agarose gels: Effect of agarose content and modeling

“…However, the quantitative interpretation is often complicated by artifacts arising from the different adsorptive behavior of native and fluorescently labeled protein molecules and by fluorescence attenuation effects. 9,16 Refractive index microscopy, as recently introduced, 11,12 allows measurements with unlabeled proteins but is restricted to cases where the concentration profile is very sharp. Finally, light microscopy with gels supported in square capillaries can provide quantitative insight, but requires special syntheses and is cumbersome when the gel is produced through a multistep process, such as is the case for charged agarose gels.…”