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. 1997 Aug 4;186(3):393–403. doi: 10.1084/jem.186.3.393

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Redox conditions determine the proper formation of intra- and intermolecular disulfide bonds in vitro. (A) Formation of intrachain disulfide bridges and proper folding of TCR-β and CD4 is dependent on an oxidizing environment; TCR-β and CD4 were translated for 1 h at 30°C in the presence of DTT and GSSG at the indicated concentrations. Immunoprecipitations were performed with the indicated conformation-specific antibodies after lysis of the microsomes in NP-40 lysis buffer in the presence of 50 mM iodoacetamide. Total microsome-associated material as well as immunoprecipitates were analyzed by SDS-PAGE (12.5%), performed under nonreducing conditions. (B) The formation of disulfide-linked ζ dimers occurs under proper redox conditions: TCR-ζ was translated for 1 h at 30°C in the presence of DTT and GSSG at indicated concentrations. Lysates were prepared in NP-40 lysis buffer in the presence of 50 mM iodoacetamide and subjected to immunoprecipitation using the mAb TIA-2. The ζ homodimer migrates in SDS-PAGE (12.5%) performed under nonreducing as a complex with a molecular weight of 30 kD but is converted to monomeric TCR-ζ if analyzed by reducing SDS-PAGE. Total microsome-associated material was analyzed by reducing SDS-PAGE and serves as a control for the quantity of TCR-ζ expressed.

Redox conditions determine the proper formation of intra- and intermolecular disulfide bonds in vitro. (A) Formation of intrachain disulfide bridges and proper folding of TCR-β and CD4 is dependent on an oxidizing environment; TCR-β and CD4 were translated for 1 h at 30°C in the presence of DTT and GSSG at the indicated concentrations. Immunoprecipitations were performed with the indicated conformation-specific antibodies after lysis of the microsomes in NP-40 lysis buffer in the presence of 50 mM iodoacetamide. Total microsome-associated material as well as immunoprecipitates were analyzed by SDS-PAGE (12.5%), performed under nonreducing conditions. (B) The formation of disulfide-linked ζ dimers occurs under proper redox conditions: TCR-ζ was translated for 1 h at 30°C in the presence of DTT and GSSG at indicated concentrations. Lysates were prepared in NP-40 lysis buffer in the presence of 50 mM iodoacetamide and subjected to immunoprecipitation using the mAb TIA-2. The ζ homodimer migrates in SDS-PAGE (12.5%) performed under nonreducing as a complex with a molecular weight of 30 kD but is converted to monomeric TCR-ζ if analyzed by reducing SDS-PAGE. Total microsome-associated material was analyzed by reducing SDS-PAGE and serves as a control for the quantity of TCR-ζ expressed.