Protein-labeling effects in confocal laser scanning microscopy.

The journal of physical chemistry. B (2006-07-21)
Christopher A Teske, Magnus Schroeder, Robert Simon, Jürgen Hubbuch
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 520-Biotin, BioReagent, suitable for fluorescence, ≥95.0% (HPCE)
Supelco
Atto 520 NHS ester, BioReagent, suitable for fluorescence, ≥80.0% (coupling to amines)
Sigma-Aldrich
Atto 520 azide, suitable for fluorescence
Sigma-Aldrich
Atto 520 maleimide, for fluorescence, ≥90% (coupling rate)

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