Eur Biophys J DOI 10.1007/s00249-008-0291-2 123 ORIGINAL PAPER Monitoring the assembly of antibody-binding membrane protein arrays using polarised neutron reXection Anton P. Le Brun · Stephen A. Holt · Deepan S. Shah · Charles F. Majkrzak · Jeremy H. Lakey Received: 29 September 2007 / Revised: 1 February 2008 / Accepted: 11 February 2008  EBSA 2008 Abstract Protein arrays are used in a wide range of appli- cations. The array described here binds IgG antibodies, pro- duced in rabbit, to gold surfaces via a scaVold protein. The scaVold protein is a fusion of the monomeric E. coli porin outer membrane protein A (OmpA) and the Z domain of Staphylococcus aureus protein A. The OmpA binds to gold surfaces via a cysteine residue in a periplasmic turn and the Z domain binds immunoglobulins via their constant region. Polarised Neutron ReXection is used to probe the structure perpendicular to the gold surface at each stage of the assembly of the arrays. Polarised neutrons are used as this provides a means of achieving extra contrast in samples having a magnetic metal layer under the gold surface. This contrast is attained without resorting to hydrogen/deute- rium exchange in the biological layer. Polarised Neutron ReXection allows for the modelling of many and complex layers with good Wts. The total thickness of the biological layer immobilised on the gold surface is found to be 187 Å and the layer can thus far be separated into its lipid, protein and solvent parts. Abbreviations BME 2-Mercaptoethanol DTT Dithiothreitol IgG Immunoglobulin G IMAC Immobilized metal aYnity chromatography IPTG Isopropyl--D-thiogalactopyranoside OG n-Octylglucoside OmpA Outer membrane protein A OmpAZ Circularly permuted OmpA with two SpA Z domains fused at the N terminus PNR Polarised Neutron ReXection Q Momentum transfer SDS Sodium dodecyl sulphate SLD Scattering length density SpA Staphylococcus aureus protein A TCEP Tris(2-carboxy-ethyl)phosphine hydrochloride thioPEG Polyethylene glycol with a thiolalkane func- tional group Introduction Protein arrays Protein arrays are becoming important tools for various applications including: use as scaVolds for tissue engineer- ing (Liu et al. 2007), proteomics (Unwin et al. 2006) and point of use diagnostics (Kingsmore 2006). Unlike nucleic Advanced neutron scattering and complementary techniques to study biological systems. Contributions from the meetings, ‘‘Neutrons in Biology’’, STFC Rutherford Appleton Laboratory, Didcot, UK, 11–13 July and ‘‘Proteins At Work 2007’’, Perugia, Italy, 28–30 May 2007. A. P. Le Brun · J. H. Lakey (&) Institute for Cell and Molecular Biosciences, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK e-mail: j.h.lakey@ncl.ac.uk A. P. Le Brun e-mail: anton.le-brun@ncl.ac.uk S. A. Holt ISIS, STFC Rutherford Appleton Laboratory, Didcot, Oxfordshire OX11 0QX, UK D. S. Shah Orla Protein Technologies Ltd, Nanotechnology Centre, Herschel Building, Newcastle upon Tyne NE1 7RU, UK C. F. Majkrzak NIST Centre for Neutron Research, Gaithersburg, MD 20899, USA