Analysis of the molecular organization of recombinant human tumor necrosis factor (rTNF) in solution using ethylene glycolbis(succinimidylsuccinate) as the cross-linking reagent.
Analysis of chemically cross-linked recombinant human tumor necrosis factor (rTNF) on sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the presence of three distinct molecular forms which correspond to trimers, dimers, and monomers. The cross-linking procedure appeared to enhance the antigenicity of these molecular forms of rTNF as determined by Western blot analysis. Cross-linking was unaffected by 2% Nonidet P-40, high concentrations of human serum albumin, or 60% normal human serum. Sodium dodecyl sulfate (0.08%) completely abolished the cross-linking of rTNF. This paper demonstrates that rTNF, at a concentration as low as 0.8 ng/ml, exists solely in the trimeric form in solution under nondenaturing conditions.
Lam
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KS
Arizona Cancer Center, University of Arizona College of Medicine, Tucson 85724.
Scuderi
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Salmon
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D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D011994:Recombinant Proteins; D012996:Solutions; D013388:Succinimides; D014409:Tumor Necrosis Factor-alpha
D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D012996:Solutions; D013388:Succinimides; D014409:Tumor Necrosis Factor-alpha; C028722:ethylene glycolylbis(succinimidyl succinate)