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. 2011 Mar 14;50(12):2761-3.
doi: 10.1002/anie.201007626. Epub 2011 Mar 2.

The binding of fluorophores to proteins depends on the cellular environment

Yun Kyung Kim  1 , Jun-Seok Lee, Xuezhi Bi, Hyung-Ho Ha, Shin Hui Ng, Young-hoon Ahn, Jae-Jung Lee, Bridget K Wagner, Paul A Clemons, Young-Tae Chang
Affiliations

The binding of fluorophores to proteins depends on the cellular environment

Yun Kyung Kim et al. Angew Chem Int Ed Engl. .
No abstract available

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Figures

Figure 1
Figure 1. Probes for myogenic differentiation
(a) Screen of rosamine library. Myoblasts or myotubes were incubated with 500 nM of library compounds for 2 hours before imaged (b) Chemical structures of selected probes, I25 and I31. (c) Fluorescent images of I25 and I31 before (right) and after (left) muscle differentiation. Scale bar = 20 µm.
Figure 2
Figure 2. Identification of protein-binders in vitro vs. in living cells
(a) Chemical structure of the affinity matrix used to isolate cellular proteins. (b) SDS-PAGE analysis of bead-bound proteins from C2C12 myotubes. In a competition assay, myotube lysates were pre-incubated with 100 µM of I25, I31, or control compounds (rhodamine 123 and rhodamine B) before the affinity pull-down experiment. (c) Chemical structure of CDy2 for labeling target protein in living cells. (d) Myoblasts (MB) or myotubes (MT) were incubated with CDy2 (500 nM) for 30 min and imaged with a fluorescent microscope. Then, cells were lysed for in-gel fluorescence analysis (λex = 530 nm, λex = 580 nm) (e). (f) 2D-gel analysis for the identification of labeled-protein.
Figure 3
Figure 3. Validation of labeled protein identity in living cells
(a) GFP-tagged ALDH2 or tubulin constructs were transfected into HEK293 cells. After 48 hours, cells were labeled with CDy2 and subjected to in-gel fluorescence imaging. Immunoblot analysis shows the endogenous (black arrow) and over-expressed (red arrow) proteins. (b) C2C12 myoblasts were transfected with siRNA against ALDH2. After 72 hours of transfection, cells were labeled with CDy2. Fluorescence labeling patterns of CDy2 were directly compared to ALDH2 immunofluororescence (green).
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