Figure 1

In vivo conjugation of QD's to Akt-PH-EGFP via intein mediated protein splicing. (a) Schematic representation of site-specific intein-mediated conjugation of QD's to target protein. (b) Co-localization of QDot₅₈₅ with Akt-PH-EGFP on the cell membrane. Fluorescence images of stage 10 Xenopus embryos microinjected with the probe (I_C-QDot₅₈₅) shown in red, in one blastomere at the two-cell stage, and then injected with RNA encoding the target protein (Akt PH-EGFP-I_N) shown in green, in three of four blastomeres. Yellow shows the overlap between red QDot₅₈₅ and green EGFP indicating successful QD-protein conjugation in a live embryo. (c) Biochemical characterization of protein-QD conjugates. Xenopus embryos were injected with either probe (Ic-QD's) only or Btk-PH-EGFP-I_N RNA only or both, lysed at stage 10 and loaded onto a 0.5% agarose gel. QDot₆₅₅ were visualized with a band pass 650/30 emission filter under UV excitation and GFP was imaged with a band pass 500/50 filter set on UVP iBox Imaging System. The ligation product appears as a smeary band under both the GFP and QD filters, only in lysates of Xenopus embryos injected with both the RNA and the probe, and is denoted as Btk-PH-EGFP-QD conjugate. A single band corresponding to the Btk-PH-EGFP protein fusion that is not conjugated to QD's is also detectable under the GFP filter, in lysates of Xenopus embryos injected with RNA only or RNA and probe, but not QD's only.