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Separation of tumor necrosis factor α isoforms by two‐dimensional polyacrylamide gel electrophoresis
Author(s) -
Watts Alan D.,
Hunt Nicholas H.,
Hambly Brett D.,
Chaudhri Geeta
Publication year - 1997
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.1150180710
Subject(s) - tunicamycin , gel electrophoresis , polyacrylamide gel electrophoresis , microbiology and biotechnology , glycosylation , gene isoform , transmembrane protein , isoelectric focusing , blot , chemistry , biochemistry , tumor necrosis factor alpha , biology , enzyme , endoplasmic reticulum , gene , unfolded protein response , immunology , receptor
The mouse macrophage cell‐line RAW264.7, stimulated with lipopolysaccharide, was used as a model for the study of the production of tumor necrosis factor (TNF) isoforms. TNF is synthesised initially as a 26 kDa transmembrane precursor, which is then processed enzymatically by a protease to release a mature molecule of 17 kDa. Dose‐dependent production of transmembrane TNF was assessed by fractionation of cell membranes and Western blot analysis followed by autoradiography and densitometry. Isoforms of both the precursor and mature molecules were separated using two‐dimensional (2‐D) electrophoresis with immobilised pH gradient 3–10 linear gels as the first dimension. After radiolabelling of cells with 35 S, both cell‐associated and supernate‐associated TNF isoforms were immunoprecipitated. A large number of protein spots were visualised on the 2‐D gel map, for both the transmembrane and mature TNF species, more than have been detected previously using one‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The likelihood that these putative isoforms were the result of differential glycosylation was tested by preincubating the cells with tunicamycin. This had the effect of reducing the number of protein spots, notably the higher molecular weight species. There were a number of precursor TNF isoforms that were unchanged upon tunicamycin treatment and these presumably reflect protein modifications other than glycosylation.