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. 2016 Jun 30;6:29130. doi: 10.1038/srep29130

Figure 2. Determination of the distance between N- and C-termini of GLUT1 using BRET.

Figure 2

(a) Cartoon diagrams of the vectors used for determination of BRET values for dual-labeled GLUT1. The background BRET ratio (610 nm/410 nm signal) was determined using GLUT1 fused to Nluc alone, while a dual-tagged vector with mCherry linked to the N-terminus and Nluc linked to the C-terminus was used to measure the average distance between these two sites. (b) Absolute BRET ratios are shown for each of the indicated fusion constructs, with error bars representing standard deviations of triplicate experiments. The BRET ratio for the dual-tagged GLUT1 lies within the range of values determined for the linker vector series. (c) The linker vector series was used to create a calibration curve relating distance to linker number, and therefore BRET ratio. Fitting of the data derived from the dual-tagged GLUT1 vector to this curve produced an estimated distance of 28.28 +/− 7.61 angstroms between the N- and C-termini of GLUT1.