Agilent Attaching Antibodies to AFM Probes with Sulfhydryl Reactive PEG Tether NHS-PEG18-PDP - App Note 5989 free download

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Keysight Technologies Attaching Antibodies to AFM Probes with the Sulfhydryl Reactive PEG Tether, NHS-PEG18-PDP Application Note Introduction AFM probes can be transformed into sensitive, chemically selective biosensors by attaching ligand molecules to the tips of the probes. Single-molecule molecular recognition force microscopy (MRFM) is an AFM-based technique that relies heavily on probes that have been modified with ligand molecules [Riener et al. 2003, Hinterdorfer 2004]. In MRFM, single-molecule unbinding interactions between ligands and complementary receptors that are immobilized on a substrate are observed and quantified one by one as the AFM cantilever approaches and subsequently withdraws from the substrate. These force spectroscopy (FS) experiments can provide valuable information about the structure and dynamics of molecular unbinding events at the single-molecule level [Noy et al. 1997]. The technique has also been effectively applied to gain an understanding of the intramolecular forces involved in protein folding and polymer elongation [Allison et al. 2002]. Topography and recognition imaging (TREC) is another single-molecule AFM technique that utilizes ligands on an AFM probe and complementary receptors on a substrate. TREC resolves recognition maps of ligand-receptor interactions by scanning an AFM probe, which contains immobilized ligands, over a substrate, which contains receptors, in magnetic AC (MAC) Mode. Using a Keysight AFM equipped with PicoTREC, which resolves the TREC AFM signals, the lateral positions of functionally active receptors can be resolved with nanometer resolution [Stroh et al. 2004a, Stroh et al. 2004b, Kienberger et al. 2004b]. PicoTREC has been used to image, map, and analyze the chemical compositions of a variety of samples, including molecular interactions between nucleic acids and proteins [Lin et al. 2006], antibodies and antigens [Marcus et al. 2006, Stroh et al. 2004a, Stroh et al. 2004b], and small ligands and their receptors [Ebner et al. 2005]. There are many biochemical immobilization and bioconjugation chemistry schemes that have been applied to the investigation of ligand-receptor interactions by MRFM and TREC imaging. In such studies, biological ligands are typically bound to the tip of an AFM probe, such as a MAC lever, while corresponding receptor molecules are bound to a flat substrate, such as mica, silicon, flat glass, or a gold-coated substrate. This protocol describes the attachment of antibodies to AFM probes via short polyethylene glycol (PEG) linkers. The heterobifunctional, amine and sulfhydryl reactive PEG linker, PDP-PEG18-NHS, should be synthesized in an organic chemistry laboratory [Haselgruber et al. 1995, Kamruzzahan et al. 2006] or purchased from a vendor that performs custom synthesis. The AFM probes will be cleaned, activated with an alkoxy aminosilane, PEGylated with PDP-PEG18-NHS, and then conjugated with the antibody. The antibo