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. Author manuscript; available in PMC: 2020 Mar 23.
Published in final edited form as: Methods. 2018 Nov 10;157:66–79. doi: 10.1016/j.ymeth.2018.11.003

Fig. 2.

Fig. 2.

Evaluation of auto-biotinylation by mutants of BirA at position 118. (A) Purification and biotinylation state (fl-neutra detection) of wild-type and mutant BirA proteins expressed as MBP fusions in E. coli. Each mutant protein contains a single amino acid substitution at position 118, as indicated. Equal amounts (1 μg) of each MBP-BirA mutant were analyzed by Coomassie blue staining and by blotting. (B) Scheme for Factor Xa mediated cleavage and analysis of auto-biotinylation. (C) Biotin detection within the MBP and BirA catalytic fragments of each BirA mutant (2.5 μg) before and after cleavage with Factor Xa. Blots were also probed with anti-MBP antibody. Bands corresponding to MBP-BirA, MBP, and BirA are indicated to the right of each panel. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)