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. 2001 Feb;183(4):1346–1358. doi: 10.1128/JB.183.4.1346-1358.2001

FIG. 5.

FIG. 5

(A) Western blot assay carried out in bacterial lysates to detect cytoplasmic formation of disulfide bonds in the tHG-93CTPB hybrid expressed in a trxB mutant. All E. coli AD494 and DH5α strains carrying both ptGH-93CTPB and pRUW4 plasmids were grown overnight and induced with 1 mM IPTG for 3 h. Bacterial cells (1 ml) were centrifuged in a microcentrifuge, and pellets were lysed by boiling for 5 min in 100 μl of Laemmli sample buffer (40). Aliquots of 30 μl were loaded in an SDS–12% polyacrylamide gel. Lanes: Std, GIBCO-BRL molecular mass standard; 1, DH5α/ptGH-93CTPB plus 25 mM β-ME; 2, DH5α/ptGH-93CTPB without β-ME; 3, AD494/ptGH-93CTPB plus 25 mM β-ME; 4, AD494/ptGH-93CTPB without β-ME. (B) Detection of change in electrophoretic mobility of the tGH-181CTPB hybrid in DH5α cell extracts after treatment with β-ME. Proteins were resolved by 8 M urea–8% polyacrylamide gel electrophoresis (see Materials and Methods), and the hybrid (arrows) was detected by Western blotting as described in Materials and Methods. Lanes: 1, ptGH-181CTPB without β-ME; 2, ptGH-181CTPB with 10 mM β-ME; 3, ptGH-181CTPB plus pRUW4, without β-ME added; 4, ptGH-181CTPB plus pRUW4 with 10 mM β-ME. Loaded samples contained 10 μl of cell extracts and 10 μl of Laemmli sample buffer without SDS or β-ME. Hybrids loaded on lanes 1 and 3 did not enter the gel and were lost prior to or during transfer to the nitrocellulose filter. (C) Detection of change in electrophoretic mobility of hEPO-181 hybrid in DH5α cells overexpressing the dsbC gene by β-ME treatment of extracts previously blocked with 20 mM iodoacetic acid. Reaction with iodoacetic acid was done as described in Materials and Methods. Proteins were resolved by 8 M urea–8% polyacrylamide gel electrophoresis, and the hybrid (arrows) was detected by a chemiluminescence assay (see Materials and Methods). Loaded samples contained 10 μl of cell extracts and 10 μl of Laemmli sample buffer without SDS or β-ME. Lanes: 1, phEPO-181CTPB plus pRUW4 plus pACYC-dsbC, without β-ME added; 2, phEPO-181CTPB plus pRUW4 plus pACYC-dsbC with 10 mM β-ME.