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Phospholipids as an alternative to direct covalent coupling: surface functionalization of nanoporous alumina for protein recognition and purification.

Journal of colloid and interface science (2011-10-29)
Thomas D Lazzara, Daniela Behn, Torben-Tobias Kliesch, Andreas Janshoff, Claudia Steinem
ABSTRACT

Anodic aluminum oxide (AAO) substrates with aligned, cylindrical, non-intersecting pores with diameters of 75 nm and depths of 3.5 or 10 μm were functionalized with lipid monolayers harboring different receptor lipids. AAO was first functionalized with dodecyl-trichlorosilane, followed by fusion of small unilamellar vesicles (SUVs) forming a lipid monolayer. The SUVs' lipid composition was transferred onto the AAO surface, allowing us to control the surface receptor density. Owing to the optical transparency of the AAO, the overall vesicle spreading process and subsequent protein binding to the receptor-doped lipid monolayers could be investigated in situ by optical waveguide spectroscopy (OWS). SUV spreading occurred at the pore-rim interface, followed by lateral diffusion of lipids within the pore-interior surface until homogeneous coverage was achieved with a lipid monolayer. The functionality of the system was demonstrated through streptavidin binding onto a biotin-DOPE containing POPC membrane, showing maximum protein coverage at 10 mol% of biotin-DOPE. The system enabled us to monitor in real-time the selective extraction of two histidine-tagged proteins, PIGEA14 (14 kDa) and ezrin (70 kDa), directly from cell lysate solutions using a DOGS-NTA(Ni)/DOPC (1:9) membrane. The purification process including protein binding and elution was monitored by OWS and confirmed by SDS-PAGE.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 633, BioReagent, suitable for fluorescence
Supelco
Atto 633 NHS ester, BioReagent, suitable for fluorescence, ≥90.0% (coupling to amines)
Sigma-Aldrich
Atto 633 amine, suitable for fluorescence
Sigma-Aldrich
Atto 633 iodoacetamide, suitable for fluorescence
Sigma-Aldrich
Atto 633 azide, suitable for fluorescence, ≥80.0% (HPCE)

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