Confocal microscopy as a tool for studying protein adsorption to chromatographic matrices

Confocal scanning laser microscopy was used for studying protein adsorption to affinity chromatography matrices. The adsorption of Protein A to IgG Sepharose 6 Fast Flow was studied by batch incubation with varying amounts of fluorescently labeled Protein A. At low sample amounts, Protein A had been adsorbed to a thin outer layer. By increasing the sample-IgG Sepharose ratio, the adsorption layer also increased. Likewise, the adsorption depth was dependent on the incubation time. Finally, a stack of confocal images separated in space was used for three-dimensional reconstruction of the adsorption pattern in a particle.