Electrophoretic properties of human IgG and its subclasses on sodium dodecyl-sulfate-polyacrylamide gel electrophoresis and immunoblots.
Unreduced human immunoglobulin G (IgG) which was not aggregated showed anomalous apparent molecular masses on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It migrated mainly as three distinct bands with apparent molecular masses from 190 to 240 kDa on gels containing 8% polyacrylamide, when denatured at 37 degrees C. Generation of this banding pattern has two reasons: (a) the pattern is a superposition of bands originating from the four IgG subclasses that differ in molecular masses and structures; and (b) the complexity of the band pattern is further increased, because IgG myeloma proteins of the IgG1 and IgG2 subclass migrated as doublets, while IgG3 and IgG4 formed primarily one band with slightly different apparent molecular masses. These properties were independent of the type of light chain in all myeloma proteins studied. Generation of doublets suggests heterogeneities of monoclonal proteins. The two separable protein populations from IgG1 differ in their susceptibility to reduction. Reduction at 37 degrees C cleaved the larger into heavy and light chain, while it generated heavy chain dimer and light chain from the smaller species. Hence, it is possible that monoclonal IgG1 are comprised of at least two subpopulations of molecules with different S-S bonds. Doublet formation of IgG2 remains unexplained, since both species were equally sensitive to reduction. Knowledge on the anomalous properties of IgG on SDS-PAGE is a prerequisite to run immunoblots from unreduced cellular antigens without confounding cell-associated IgG with cellular antigens.
Fasler
S
S
Department of Biochemistry, Swiss Federal Institute of Technology, ETH-Zentrum, Zurich.
Skvaril
F
F
Lutz
H U
HU
D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007074:Immunoglobulin G / Q000145:classification / Q000302:isolation & purification*; D009194:Myeloma Proteins / Q000032:analysis / Q000145:classification; D012967:Sodium Dodecyl Sulfate
D007074:Immunoglobulin G; D009194:Myeloma Proteins; D012967:Sodium Dodecyl Sulfate