Peter Wagner

Optimizing antibody immobilization strategies for the construction of protein microarrays

Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the capture agent). This in turn is a function of the surface density and fractional activity of the capture agents. Here we investigate how these two factors are affected by the orientation of the capture agents on the surface. We compare randomly versus specifically oriented capture agents based on both full-sized antibodies and Fab fragments. Each comparison was performed using three different antibodies and two types of streptavidin-coated monolayer surfaces. The specific orientation of capture agents consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvements over surfaces with randomly oriented capture agents. Surface plasmon resonance revealed a dense monolayer of Fab fragments that are on average 90% active when specifically oriented. Randomly attached Fabs could not be packed at such a high density and generally also had a lower specific activity. These results emphasize the importance of attaching proteins to surfaces such that their binding sites are oriented toward the solution phase.

Immobilization strategies for biological scanning probe microscopy1

Biological atomic force microscopy (AFM) is now established as a method for studying the structure and function of biomolecular objects at the solid‐liquid interface. Major progress in this field is linked to new developments in instrumentation, a better understanding of tip‐sample interactions, and improved sample preparation techniques. In this review, the most common strategies for biomolecular immobilization with respect to biological AFM applications are summarized.

Reversible, site-specific immobilization of polyarginine-tagged fusion proteins on mica surfaces.

A large variety of genes is expressed as fusion proteins for the purpose of characterization and purification in molecular biology. We have used this strategy to append polyarginine peptides in order to achieve specific binding of the Arg‐tag to atomically flat, negatively charged mica surfaces. We show that the model protein, hexaarginine‐tagged green fluorescent protein (GFP), binds to mica via its Arg‐tag based on ion exchange of naturally occurring potassium cations. Only non‐specific binding was observed with the control protein that is free of the Arg‐tag. This novel technology will be widely applicable to orient functional proteins on flat surfaces.

Light-harvesting complex II in monocomponent and mixed lipid-protein monolayers

Monomolecular layers at the air-water interface were formed directly with isolated largest light-harvesting pigment-protein complex of Photosystem II (LHC II) or out of egg yolk lecithin (EYL) liposomes containing incorporated LHC II. Pure protein monolayers showed a mean area of 1400 A2 per molecule at the air-water interface. Monolayers were deposited onto glass slides by means of Langmuir-Blodgett (LB) technique. Chlorophyll fluorescence of LHC II-LB and EYL-LHC II-LB films proved energetic coupling of chlorophyll a and b, thus indicating native conformation of LHC II within the monolayers. Scanning force microscopy (SFM) revealed ring-like structures formed in monocomponent protein layers as well as in mixed protein-lipid films. These results suggest that a structural arrangement of LHC II is favoured in a lipid environment but that the protein has itself a strong tendency for structural complex rearrangement in our system.