Concentration and separation of low-abundant proteins in human plasma by ammonium sulfate fractionation followed by a three-step electrophoresis technique

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  • Mukai Jun
    Department of Chemistry, Faculty of Science, Ehime University
  • Manabe Takashi
    Department of Chemistry, Faculty of Science, Ehime University

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Abstract

In this paper, we describe a technique to concentrate and separate low-abundant proteins in human plasma. A human plasma sample was fractionated by ammonium sulfate and the precipitate obtained at 35% saturated ammonium sulfate was dialyzed and subjected to isoelectric focusing employing an agarose slab gel (55mm×73mm×3mm, 1% agarose). The IEF gel was cut into strips and each strip was transferred on the top of an SDS-containing column gel (5% T, 5% C polyacrylamide gel) prepared in a Pasture pipette. After SDS electrophoresis, each pipette column gel was transferred onto a top of a micro slab gel (38mm×38mm×1mm, 8-17% T, 5% C polyacrylamide gradient gel) and second SDS gel electrophoresis was run. If the step of pipette electrophoresis was deleted, the proteins appeared as horizontal faint bands on the slab gel. By using the three-step electrophoresis, the low-abundant proteins were detected as densely stained spots on the slab gel with 10-25 fold higher concentration of corresponding proteins than in the case of two-step electrophoresis. The spots on the slab gels after the three-step electrophoresis have been subjected to protein identification by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and peptide sequencing using electrospray ionization tandem mass spectrometry (ESI-MS/MS). The results showed the advantages of the present technique for the structural analysis of low-abundant proteins in human plasma.

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