Protocol abstract
Nature Protocols 2, - 1907 - 1917 (2007)
Published online: 2 August 2007 | doi:10.1038/nprot.2007.262
Subject Category: Biochemistry and protein analysis
Frontal affinity chromatography—mass spectrometry
Ella S M Ng1, Nora WC Chan2, Darren F Lewis3, Ole Hindsgaul4 & David C Schriemer1
Abstract
Frontal affinity chromatography (FAC) is a biophysical method for the discovery and characterization of molecular interactions in a flow-based system. Several different modes of analysis are possible by interfacing to the mass spectrometer, including robust single-compound characterizations as well as high-throughput screening of over 1,000 compounds per run. The method supports thermodynamic and kinetic characterization of interactions for a wide range of molecular species and possesses similarities to flow-based biosensors such as surface plasmon resonance. It offers sensitive detection of ligands present well below their respective dissociation constants, and can be assembled from readily available laboratory components. Direct coupling of the FAC cartridge to the mass spectrometer is useful for the interrogation of single compounds or mixtures of limited complexity. An offline fractionation schema is more appropriate for discovery-mode applications. A high-performance FAC system enabling both modes can be assembled in 2–3 h. Measurements of dissociation constants can be made with such a system in 0.5–3 h, and the system supports higher-throughput screening modes at a rate of 10,000 compounds d- 1.
- Department of Biochemistry and Molecular Biology, University of Calgary, Calgary T2N 4N1 Alberta, Canada.
- Defence R & D Canada—Suffield, P.O. Box 4000, Medicine Hat T1A 8K6 Alberta, Canada.
- Upchurch Scientific Inc., IDEX Health & Science, 619 Oak Street, Oak Harbor, Washington, USA.
- Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen-Valby, Denmark.
Correspondence to: David C Schriemer1 e-mail: dschriem@ucalgary.ca
nature-products
A-Z product listing
- ammonium acetate(Sigma)
- Breuchbuler packing bomb(Breuchbuhler)
- Empty cartridges holders(Upchurch Scientific)
- EZ-link Sulfo-NHS-LC-biotin(Pierce)
- Gastight syringes(Hamilton Inc)
- Gradient NFM(Eksigent)
