Structural analyses of proteins electroblotted from native polyacrylamide gels onto polyvinyldiene difluoride membranes. A method for determining the stoichiometry of protein-protein interaction in solution.
The usefulness of a native gel electroblotting technique in the study of protein-protein interactions was demonstrated by the determination of the stoichiometry of the interaction between interleukin-2 (IL-2) and the alpha subunit of IL-2 receptor (IL-2R alpha) in solution. Complexes formed between the recombinant forms of the two proteins in solution were separated from the noncomplexed protein molecules by electrophoresis in a native polyacrylamide gel and the protein bands were electroblotted quantitatively onto polyvinyldiene difluoride membranes for further structural analysis. The data obtained from sequence and amino acid analyses of the blotted proteins provided direct evidence that IL-2 binds to IL-2R alpha in a 1:1 ratio. This methodology should be applicable to the study of other structure/function aspects of protein-protein interactions in solution.
Wang
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Department of Protein Biochemistry, Hoffmann-La Roche Incorporated, Nutley, New Jersey 07110.
Pan
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YC
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D007376:Interleukin-2 / Q000737:chemistry* / Q000302:isolation & purification; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D011145:Polyvinyls; D015375:Receptors, Interleukin-2 / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate; D012996:Solutions; D013329:Structure-Activity Relationship; D005465:Fluorocarbon Polymers
D000596:Amino Acids; D007376:Interleukin-2; D008567:Membranes, Artificial; D011145:Polyvinyls; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D012996:Solutions; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers