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. 2012 May 31;7(5):e37598. doi: 10.1371/journal.pone.0037598

Figure 6. S-CROSS assay for the detection of ligand-receptor interactions.

Figure 6

(A) Scheme of the S-CROSS assay. SNAP-receptor and ligand-SNAP-CLIP are incubated to form a complex. BG-fluorophore-BC preferentially crosslinks proteins that are in close spatial proximity. (B) SNAP-eDHFR WT (1 µM) was mixed with MTX-SNAP-CLIP or unlabeled SNAP-CLIP (1 µM) in the presence or absence of free MTX (50 µM). The experiments were performed in the absence or presence of 100 µM NADPH. Then, the mixture was treated with 2.5 µM BG-647-BC. Labeled proteins were resolved by SDS-PAGE and detected by in-gel fluorescence scanning. (C) Fluorescence signal of the hetero-crosslinking products in the presence or absence of free MTX (Mean±SD, n = 3–5). # represents P = 0.001 in paired t-test. (D) S-CROSS assay in cell extract. SNAP-ORP7 was expressed in HEK293 cells. After preparation of extract, SNAP-ORP7 was subjected to S-CROSS assay using erlotinib-SNAP-CLIP (2 µM) in the absence or presence of free erlotinib (10 µM).