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. Author manuscript; available in PMC: 2017 Nov 15.
Published in final edited form as: Methods Mol Biol. 2016;1461:117–130. doi: 10.1007/978-1-4939-3813-1_9

Fig. 1. pH response of purified pHlash protein in vitro.

Fig. 1

(A) SDS-PAGE gel of purified His-tagged pHlash protein stained with Coomassie Blue dye. Leftmost lane is molecular weight standards with KDa indicated, while the other lanes are the purified pHlash protein loaded at 0.2, 1, and 2 μg per lane. (B) Construct of the pHlash fusion protein. Rluc8 was linked to cpVenus by the sequence Ala-Glu-Leu. (C) Raw data (not normalized) of luminescence emission spectra of purified pHlash protein with 10 μM native coelenterazine at different pH values (pH 5.4-9.0) in 50 mM BIS-Tris-propane, 100 mM KCl, and 100 mM NaCl. (D) Normalized luminescence emission spectra of pHlash measured as in panel C. Luminescence intensity was normalized to the peak at 475 nm. (E) The BRET ratio (luminescence at 525nm:475 nm) as a function of pH is shown for pHlash in vitro. Error bars are +/- S.D., but in most cases the error bars are so small that they are obscured by the symbols (n = 3).