Journal: Bioconjugate Chemistry
Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors
Figure Lengend Snippet: Characterization of probes binding on A431 cell surface. (A) Dissociation constant analysis of probes on cell surface. 5 × 10 5 /mL quantities of cells were incubated with probes for 1 h at 37 °C followed by 2 μM MG-B-tau for 5 min. Then cells were kept on ice for flow cytometry. The mean fluorescence intensity was corrected with background of cells incubating with F/MG and then normalized to mean fluorescence at 250 nM of probes. (B) Competition assay of nonfluorescent affibody A binding to the cell surface. Cells were labeled with 250 nM AFA or F and a serial dilution of A followed by 100 nM of MG-B-tau added prior to measurement. (C) Detection of receptor activation by Western blots. Starved cells were labeled with 250 nM probes followed by 100 nM of MG-B-tau and then cells were treated with 100 ng/mL EGF. Then cells were lysed for Western blot in order to detect phosphorylated EGFR and total EGFR. (D) Live-cell fluorescence microscopy of A431 cells labeled by various probes. Cells were labeled with 250 nM of probe and 100 nM of MG-B-tau prior to imaging or 100 nM of Cy5 conjugated affibody dimer. Scale bar 20 μm.
Article Snippet: A protein domain (FAP dL5** ) that binds to malachite-green (MG) derivatives for fluorescence activation was expressed as a recombinant fusion to one or two copies of the compact EGFR binding affibody ZEGFR:1907 .
Techniques: Binding Assay, Incubation, Flow Cytometry, Cytometry, Fluorescence, Competitive Binding Assay, Labeling, Serial Dilution, Activation Assay, Western Blot, Microscopy, Imaging