Fractionation enhances the resolution of proteins with similar characteristics by reducing the number of proteins that comigrate in gels, thus facilitating the detection of lower-abundance proteins and the accurate determination of quantitative and qualitative differences in disease and normal samples. An efficient, reproducible microscale fractionation protocol for complex protein mixtures using novel ion-exchange membrane chromatographic substrates (PerkinElmer, Boston, MA, USA; Vivascience, Carlsbad, CA, USA) is described. The fractionation techniques were used in combination with two-dimensional (2-D) gels and orthogonal matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify differentially expressed proteins in brain samples from persons with and without Alzheimer's disease.
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