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Differential scanning fluorimetry measurement of protein stability changes upon binding to glycosaminoglycans: a screening test for binding specificity.
Uniewicz, Katarzyna A; Ori, Alessandro; Xu, Ruoyan; Ahmed, Yassir; Wilkinson, Mark C; Fernig, David G; Yates, Edwin A.
Afiliación
  • Uniewicz KA; School of Biological Sciences, University of Liverpool, Liverpool, L69 7ZB, United Kingdom.
Anal Chem ; 82(9): 3796-802, 2010 May 01.
Article en En | MEDLINE | ID: mdl-20353159
ABSTRACT
The interaction between glycosaminoglycans (GAGs) and proteins is important for the regulation of protein transport and activity. Here we present a novel method for the measurement of protein-GAG interactions suitable for high-throughput screening, able to discriminate between the interactions of a protein with GAGs of different structures. Binding of proteins to the GAG heparin, a proxy for sulfated regions of extracellular heparan sulfate, was found to enhance the stability of three test proteins, fibroblast growth factors (FGFs)-1, -2, and -18. Chemically modified heparins and heparin oligosaccharides of different lengths stabilized the three FGFs to different extents, depending on the pattern of sugar binding specificity. The method is based on a differential scanning fluorescence approach. It uses a Sypro Orange dye, which binds to exposed core residues of a denatured protein and results in an increased fluorescence signal. It is convenient, requiring low micromolar amounts of protein and ligand compared to other interaction assays, employing only a real-time polymerase chain reaction (PCR) instrument.
Asunto(s)

Texto completo: 1 Bases de datos: MEDLINE Asunto principal: Fluorometría / Glicosaminoglicanos Tipo de estudio: Diagnostic_studies / Screening_studies Idioma: En Revista: Anal Chem Año: 2010 Tipo del documento: Article País de afiliación: Reino Unido

Texto completo: 1 Bases de datos: MEDLINE Asunto principal: Fluorometría / Glicosaminoglicanos Tipo de estudio: Diagnostic_studies / Screening_studies Idioma: En Revista: Anal Chem Año: 2010 Tipo del documento: Article País de afiliación: Reino Unido