2-DE-based proteomics for the analysis of metastasis-associated proteins.
Two-dimensional electrophoresis (2-DE) is a high-resolution technique for analysis and comparison of complex protein mixtures. With the advent of recent technical developments, its application has become significant in a wide range of fields. This chapter describes a proteomic approach for the analysis of metastasis-associated proteins using pre-fractionation of glycosylated proteins via lectin (HPA) affinity chromatography prior to separation by 2-DE. Guidelines for the preparation and storage of buffers, experimental conditions and protocols of affinity chromatography, isoelectric focussing, and SDS-PAGE conditions are provided. Critical parameters associated with the different steps of 2-DE are discussed.
Department of Molecular and Applied Biosciences, School of Life Sciences, University of Westminster, London, UK. dwekm@wmin.ac.uk
Miriam
M
Dwek
Diluka
D
Peiris
D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis* / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D037102:Lectins / Q000378:metabolism; D009362:Neoplasm Metastasis; D040901:Proteomics / Q000379:methods*
D006023:Glycoproteins; D037102:Lectins