title: Application of isothermal titration calorimetry and column chromatography for identification of biomolecular targets.
abstract: This protocol describes a method for identifying unknown target proteins from a mixture of biomolecules for a given drug or a lead compound. This method is based on a combination of chromatography and isothermal titration calorimetry (ITC) where ITC is used as a tracking tool. The first step involves the use of ITC to confirm the binding of ligand to a component in the biomolecular mixture. Subsequently, the biomolecular mixture is fractionated by chromatography, and the binding of the ligand with individual fractions (or subfractions) is verified by ITC. The iteration of chromatographic purification on the fractions combined with ITC results in identifying the target protein. This method is useful when the target protein or ligand is unknown and/or not amenable to labeling, chemical modification or immobilization. This protocol has been successfully used by our team and by others to identify both low-abundance and highly abundant target proteins present in biomolecular mixtures. With this protocol, it takes approximately 3-5 d to identify the target protein from a mixture.
authors.affiliation: Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore.
authors.forename: Xingding
authors.initials: X
authors.lastname: Zhou
authors.forename: R Manjunatha
authors.initials: RM
authors.lastname: Kini
authors.forename: J
authors.initials: J
authors.lastname: Sivaraman
mesh_terms: D001665:Binding Sites; D002151:Calorimetry / Q000379:methods*; D005591:Chemical Fractionation; D002852:Chromatography, Ion Exchange; D054501:Group II Phospholipases A2 / Q000737:chemistry* / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D030162:Reptilian Proteins / Q000737:chemistry* / Q000302:isolation & purification; D013498:Suramin / Q000737:chemistry*; D014757:Viper Venoms / Q000737:chemistry
chemical_list: D011506:Proteins; D030162:Reptilian Proteins; D014757:Viper Venoms; D013498:Suramin; D054501:Group II Phospholipases A2; C102372:ecarpholin S