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. 2005 Sep 5;390(Pt 3):655–664. doi: 10.1042/BJ20050480

Figure 2. The effect of CLC2 gene optimization on protein expression.

Figure 2

Western-blot analysis of yeast expressing N- and C-terminally GFP-tagged ClC-2. Whole cell lysates were prepared using the urea/SDS method [26], analysed by SDS/PAGE (10% polyacrylamide), transferred on to Hybond-C extra and probed with anti-GFP antibodies; equal numbers of cells were analysed. Arrow indicates the expected band of 126 kDa. (A) Yeast Δgef1 strain (RGY84) was transformed with plasmid bearing the CLC2 gene that was 5′- or 3′-terminally fused to the GFP-coding sequence (pKF16 and pKF14 respectively). Only N-terminally GFP-tagged ClC-2 was detected. The recipient strain and a strain transformed with untagged version of CLC2 (pKF20) served as controls. (B) Product of the optCLC2-GFP gene (pKF28) was detected by anti-GFP antibodies. The same recipient strain as in (A) was transformed. As a positive control the strain bearing N-terminally GFP-tagged version of ClC-2 was used. The positions of molecular-mass markers are shown alongside the gels.