Analysis of members of the Ig-gene superfamily by thermal gradient polyacrylamide gel electrophoresis.
A solid aluminum block, connected with a warm and cold thermostated waterbath, provided for a linear transversal temperature gradient (TG) during polyacrylamide gel electrophoresis (PAGE). Noncovalently bound heavy chain dimers as well as heavy-light chain dimers, derived from human monoclonal IgG, could be melted into monomers using a 40-75 degrees C TG under conditions of sodium dodecyl sulfate-PAGE. Using native PAGE, major histocompatibility complex (MHC) class I molecules, preloaded with the iodinated peptide FAPGNYPAL could be melted in a 4-40 degrees C TG to release the peptide. The method is in general applicable to thermal stability analysis of noncovalently bound hetero-oligomers if the product after melting possess different electrophoretic mobilities.
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The Netherlands Cancer Institute, Amsterdam.
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D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005803:Genes, Immunoglobulin; D015395:Histocompatibility Antigens Class I / Q000302:isolation & purification; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature
D015395:Histocompatibility Antigens Class I; D007074:Immunoglobulin G; D009842:Oligopeptides