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. Author manuscript; available in PMC: 2017 May 1.
Published in final edited form as: Protein Expr Purif. 2015 Dec 29;121:17–21. doi: 10.1016/j.pep.2015.12.001

Figure 2.

Figure 2

Anti-Gcn5 Western analysis of variant yeast Ada2Δ1/Ada3Δ3HIS/Gcn5 complexes coexpressed in E. coli. (a) Gcn5 in crude extracts (lanes 1, 3, 5, 7) or metal affinity purified (lanes, 2, 4, 6, 8) complexes coexpressed in BL21-CodonPlus(DE3)-RIL was detected using aniti-Gcn5 antibodies. The wild-type Gcn5 sequence was used in lanes 1 & 2, while the Gcn5 sequence mutated to remove the cryptic Shine-Dalgarno mutation or START codon or both elements are shown in lanes 3 & 4, lanes 5 & 6 and lanes 7&8 respectively. The truncated Gcn5 polypeptide is greatly reduced by the Shine-Dalgarno mutation, and is not detected in the START mutation sample. The position of a contaminating band that cross reacts with the anti-Gcn5 antibodies is indicated by an asterisk (*). (b) Amino acid and DNA sequences for the wild type, Shine-Dalgarno (SD), START mutation and combination Shine-Dalgarno & START mutations