Journal: PLoS ONE

Article Title: Isolation and Characterization of a Monobody with a Fibronectin Domain III Scaffold That Specifically Binds EphA2

doi: 10.1371/journal.pone.0132976

Figure Lengend Snippet: (A) FACS profiles of the original G4 library (left) and 4.5-fraction yeasts (right) obtained after final screening. Yeast cells were stained with 1 nM hEphA2 and a chicken anti-cMyc antibody. (B) Amino acid sequence alignment of monobodies and wild-type Fn3 domain. Sequences of the BC, DE, and FG loops are indicated as black boxes. (C) FACS profiles of yeasts transformed with surface display vectors, pCT-Fn3-EphA2 (E1) and pCT-Fn3-EphA2 (E10). Yeast cells were stained with 10nM hEphA2 and chicken anti-cMyc antibody.

Article Snippet: Briefly, 6.7 pmol of biotinylated hEphA2 was incubated with 10 μL of streptavidin-coated magnetic beads (Dynabeads Biotin Binder, Invitrogen, CA) in 100 μL of PBSA for 30 min at room temperature.

Techniques: Staining, Sequencing, Transformation Assay

Journal: PLoS ONE

Article Title: Isolation and Characterization of a Monobody with a Fibronectin Domain III Scaffold That Specifically Binds EphA2

doi: 10.1371/journal.pone.0132976

Figure Lengend Snippet: The indicated concentrations of hEphA2 protein were incubated with yeasts expressing each monobody. After a simple wash, yeast cells were stained with mouse anti-hEphA2 and chicken anti-cMyc antibodies, followed by staining with Alexa 488-conjugated anti-mouse and Alexa 555-conjugated anti-chicken secondary antibodies. The mean fluorescence values of each sample were measured by FACS. Affinities (Kd values) were obtained by determining the mean ratio of Alexa 488/Alexa 555 fluorescence versus hEphA2 concentration. The results are representatives of at least three independent experiments.

Article Snippet: Briefly, 6.7 pmol of biotinylated hEphA2 was incubated with 10 μL of streptavidin-coated magnetic beads (Dynabeads Biotin Binder, Invitrogen, CA) in 100 μL of PBSA for 30 min at room temperature.

Techniques: Incubation, Expressing, Staining, Fluorescence, Concentration Assay

Journal: PLoS ONE

Article Title: Isolation and Characterization of a Monobody with a Fibronectin Domain III Scaffold That Specifically Binds EphA2

doi: 10.1371/journal.pone.0132976

Figure Lengend Snippet: Streptavidin-coated magnetic beads bound with biotinylated human IgG, lysozyme, or hEphA2 were incubated with 10 nM monobody proteins with His6 (h) or AMH tags at room temperature. The bound monobodies were stained with a FITC-conjugated anti-His-tag antibody and analyzed by FACS. The results are representatives of at least three independent experiments.

Article Snippet: Briefly, 6.7 pmol of biotinylated hEphA2 was incubated with 10 μL of streptavidin-coated magnetic beads (Dynabeads Biotin Binder, Invitrogen, CA) in 100 μL of PBSA for 30 min at room temperature.

Techniques: Magnetic Beads, Incubation, Staining

Journal: PLoS ONE

Article Title: Isolation and Characterization of a Monobody with a Fibronectin Domain III Scaffold That Specifically Binds EphA2

doi: 10.1371/journal.pone.0132976

Figure Lengend Snippet: The indicated concentrations of AMH-tagged monobody proteins were incubated for 2 hr on 96-well plates coated with recombinant Eph receptor proteins. The amounts of bound monobody proteins were measured in an ELISA reader after staining with horseradish peroxidase-conjugated anti-cMyc antibodies. Data represent mean ± S.D., and asterisks (*) indicate a significant difference compared hEphA2 and other EphA series (*, P < 0.05 or **, P < 0.01). The results are representatives of at least three independent experiments.

Article Snippet: Briefly, 6.7 pmol of biotinylated hEphA2 was incubated with 10 μL of streptavidin-coated magnetic beads (Dynabeads Biotin Binder, Invitrogen, CA) in 100 μL of PBSA for 30 min at room temperature.

Techniques: Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Staining

Journal: PLoS ONE

Article Title: Isolation and Characterization of a Monobody with a Fibronectin Domain III Scaffold That Specifically Binds EphA2

doi: 10.1371/journal.pone.0132976

Figure Lengend Snippet: (A) Western blotting for hEphA2. Cell lysates were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The proteins were detected with an anti-hEphA2 antibody. (B) Surface expression of hEphA2. Cells (2 × 10 5 ) were stained with a mouse anti-hEphA2 antibody and FITC-conjugated rat anti-mouse IgG antibody. Black line, unstained cells; red line, only second Ab; blue line, anti-EphA2. (C) Quantitation of hEphA2 on the cell surface. Concentrations of hEphA2 on the cell surface were measured against an anti-rat Bangs bead standard. Data represent mean ± S.D., and asterisks (*) indicate a significant difference compared SKBR3 and PC3 cells (*, P < 0.001). (D) Measurement of E1 monobody binding to cells. Cells were incubated with 100 nM Cy5.5-conjugated E1h monobody and analyzed by FACS. Black lines, unstained cells; red lines, E1h-Cy5.5. (F) Fluorescence microscopy images of cells stained with an anti-hEphA2 antibody or E1h-Cy5.5 monobody. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Magnification of the inset with indivisual and merged images is given in the lower low. Scale bar, 20 μm. The results are representatives of at least three independent experiments.

Article Snippet: Briefly, 6.7 pmol of biotinylated hEphA2 was incubated with 10 μL of streptavidin-coated magnetic beads (Dynabeads Biotin Binder, Invitrogen, CA) in 100 μL of PBSA for 30 min at room temperature.

Techniques: Western Blot, SDS Page, Expressing, Staining, Quantitation Assay, Binding Assay, Incubation, Fluorescence, Microscopy