Automated Synthesis of Solid-Phase Bound Peptides
The SPOT method was developed by Ronald Frank for simultaneous multiple peptide synthesis on separate sites on a homogeneous membrane carrier (Frank 1992). The principle of the technique is to dispense small droplets of pre-activated amino acid derivative
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Automated Synthesis of Solid-Phase Bound Peptides HEINRICH GAUSEPOHL and CHRISTIAN BEHN
Introduction The SPOT method was developed by Ronald Frank for simultaneous multiple peptide synthesis on separate sites on a homogeneous membrane carrier (Frank 1992). The principle of the technique is to dispense small droplets of pre-activated amino acid derivatives onto a predefined array of positions on a porous membrane. The droplets are absorbed and form individual reaction compartments for chemical reaction in solid-phase synthesis.A great number of distinct spots can be arranged on a large membrane sheet and each of these is individually addressable by manual or automated delivery of the respective reagent solution. The absorptive capacity of the membrane and the volume of the drops determine the number of spots per area and the spot size. According to the specific functionality of the matrix, the spot size correlates with the particular scale of the synthesis. The synthesis is carried out using 9-fluorenyl-methoxycarbonyl (Fmoc)-protection chemistry on membranes made of pure cellulose. The amino acid building blocks are derivatives that are protected at their amino terminus by Fmoc. The protection group ensures that in each step only a single building block is coupled to each growing peptide chain. Trifunctional amino acids also carry a side-chain protection group. The Fmoc group must be removed in each synthesis cycle whereas the side-chain protection groups H. Gausepohl INTAVIS AG, Friedrich-Ebert -Strasse, 51429 Bergisch Gladbach, Germany C. Behn (~)(e-mail: [email protected], Tel.: +49-2173-89050, Fax: +49-2173-890577) ABIMED Analysen-Technik GmbH, Langenfeld, Germany Springer Lab Manual
J. Koch, M. Mahler (Eds.) Peptide Arrays on Membrane Supports © Springer-Verlag Berlin Heidelberg 2002
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HEINRICH GAUSEPOHL
and CHRISTIAN BEHN
are taken off at the end of the synthesis. In situ activation of the amino acid derivatives is performed by DIC/ HOBt, which leads to rapid activation and coupling. A membrane can be used to synthesize a large number of individual peptides, and these can be screened for biological activity by a Western-blot-like assay. Alternatively, the spots are cut out and cleaved separately f:t:om the support if a suitable linker was introduced prior to the synthesis. The SPOT method was originally developed as a manual method and steps common to all spot reactors are carried out manually by washing the whole membrane with respective reagents and solvents (see Chap. 3). The most tedious step is the spotting of individual activated amino acids onto the reactive areas in patterns, which change in each cycle. Automation of this step has made the method applicable to a much wider range of experiments, as it cuts down the amount of time involved and allows the creation of much larger arrays with smaller volumes than can be handled manually. It is now possible to synthesize several thousand peptides in a single run. Automated deposition of amino acids also allows for easy double or triple coupli
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