Munich University of Applied Sciences 1 article published in JoVE Biochemistry Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy Tanja D. Becke1,2,3, Stefan Ness2, Stefanie Sudhop1,3, Hermann E. Gaub3, Markus Hilleringmann2, Arndt F. Schilling*4, Hauke Clausen-Schaumann*1,3 1Center for Applied Tissue Engineering and Regenerative Medicine, Munich University of Applied Sciences, 2FG Protein Biochemistry & Cellular Microbiology, Munich University of Applied Sciences, 3Center for Nano Science, Ludwig-Maximilians-Universität München, 4Klinik für Unfallchirurgie, Orthopädie und Plastische Chirurgie, University Medical Center Göttingen This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.