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. Author manuscript; available in PMC: 2013 Oct 19.
Published in final edited form as: FEBS Lett. 2012 Sep 13;586(20):3716–3722. doi: 10.1016/j.febslet.2012.08.031

Fig. 4. Recoding of TAG to a sense codon.

Fig. 4

(A) BL21 and EcAR7 strains containing either a suppressor tRNA (supE) or the SEP-system (SEP) were transformed with plasmids encoding WT GFP, GFPQ94TAG, GFPQ157TAG or GFPQ94/Q157TAG, respectively. Western blots using the antibody against the N-terminus of GFP are shown (* indicates a nonspecific band). (B) EcAR7.SEP and BL21.L11C.SEP were transformed with plasmids containing GFP-variants encoding TAG at the various positions indicated. A range of site specific truncations that accumulate in BL21.L11C.SEP are indicated by a bracket (* indicates a nonspecific band). (C)EcAR7.SEP and BL21.L11C.SEP were transformed with plasmids harboring WNK4-WT and WNK4-TAG-containing variants as indicated. Western blot against the C-terminal His-tag are shown. (D) MS/MS validation of Sep insertion at E17TAG. Fragment ion spectra ([M+3H]3+ precursor = 897.452) showing characteristic neutral losses consistent with phosphoserine (SP) allowed unambiguous assignment of phosphoserine at position 17 in GFP.