Imaging the substructure of antibodies with tapping-mode AFM in air: the importance of a water layer on mica.
Monoclonal antibodies (immunoglobulin G; IgG) against the N-terminal domain of the A subunit of DNA gyrase have been imaged using tapping-mode atomic force microscopy under ambient conditions on hydrophilic mica surfaces. The familiar tri-nodal submolecular resolution of IgG (i.e. 50-kDa resolution) has been achieved when operating the microscope with the tip predominantly in the attractive force regime. Under common laboratory conditions of about 40% relative humidity, the resolution of this substructure was not achieved owing to motion of the antibodies on the surface and/or image distortion from tip-sample instabilities. Reproducible imaging of the tri-nodal antibody substructure was achieved by desiccating the samples for extended periods of time (1 week or more) before imaging. This effect is attributed to the presence of a humidity-dependent thin water layer (a few molecules or nanometres thick), which has been observed previously using the surface force apparatus and scanning polarization force microscopy. Desiccation of the mica surfaces allowed enough water to be removed from the mica surface to prevent this effect. Degradation in the image quality over the imaging period of an hour or two was observed, owing to re-adsorption of water under the ambient laboratory conditions.
Thomson
N H
NH
Department of Physics and Astronomy and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. n.h.thomson@leeds.ac.uk
D000538:Aluminum Silicates; D000911:Antibodies, Monoclonal / Q000648:ultrastructure*; D027081:DNA Gyrase / Q000276:immunology; D007074:Immunoglobulin G / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force / Q000379:methods*; D012680:Sensitivity and Specificity; D014867:Water
D000538:Aluminum Silicates; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D014867:Water; D027081:DNA Gyrase; C011934:mica