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Micropreparative two‐dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins
Author(s) -
Bjellqvist Bengt,
Sanchez JeanCharles,
Pasquali Christian,
Ravier Florence,
Paquet Nicole,
Frutiger Séverine,
Hughes Graham J.,
Hochstrasser Denis
Publication year - 1993
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501401212
Subject(s) - electrophoresis , chromatography , streaking , immobilized ph gradient , chemistry , silver stain , dimension (graph theory) , gel electrophoresis , analytical chemistry (journal) , sodium dodecyl sulfate , sample preparation , biochemistry , biology , microbiology and biotechnology , mathematics , isoelectric focusing , pure mathematics , enzyme
We describe some simple modifications to the micropreparative two‐dimensional (2‐D) electrophoresis procedure using immobilized pH gradients in the first dimension and sodium dodecyl sulfate‐electrophoresis in the second dimension. The geometry of the immobilized pH gradient strips has been changed to allow the use of large sample application cups that can accomodate greater sample volumes. The use of narrow range immobilized pH gradients with a large sample loading volume allows an efficient resolubilization of polypeptides after the first dimension. As a result, the vertical streaking caused by too high a protein concentration is eliminated in the second dimension. Protein identification by N ‐terminal sequencing is facilitated by the large protein load (1–15 mg) which can be employed using this modification. Spots not normally detectable on conventional analytical 2‐D maps, even with sensitive silver staining, are observed. Results for plasma and liver proteins are shown.