hepha2 antibody  (Santa Cruz Biotechnology)

Journal: PLoS ONE

Article Title: Engineering of monobody conjugates for human EphA2-specific optical imaging

doi: 10.1371/journal.pone.0180786

Figure Lengend Snippet: (A) Western blot analysis of hEphA2. Lysates of PC3 and HeLa cells (8 × 10 5 ) were separated by SDS-PAGE and transferred to nylon membranes. Membranes were stained with rabbit anti-hEphA2 or β-actin antibodies. (B) Flow cytometric analysis of cells. Cells (5 × 10 5 ) were stained with mouse anti-hEphA2 and Alexa 488-conjugated anti-mouse IgG antibodies (anti-hEphA2), and fluorescence was measured by flow cytometry. Unstained cells or cells stained with Alexa 488-conjugated anti-mouse IgG antibody (2 nd Ab) alone were used as controls.

Article Snippet: Cells (1 × 10 4 ) were cultured on cover glasses on 6-well plates for 24 h. To detect bound E1-Rluc8, cells were incubated with 25.5 nM E1-Rluc8 and rabbit anti-hEphA2 antibody (1:10000 dilution, Santa Cruz Biotechnology), in 3 mL of culture medium containing 3% FBS at 37°C for 2 h. After the plates were washed with 1% BSA in PBS-T, they were incubated in FITC-conjugated anti-6×His (1:1000 dilution) and Alexa 555-conjugated anti-rabbit IgG antibodies (1:1000 dilution) for 2 h in the dark at room temperature (RT).

Techniques: Western Blot, SDS Page, Staining, Fluorescence, Flow Cytometry

Journal: PLoS ONE

Article Title: Engineering of monobody conjugates for human EphA2-specific optical imaging

doi: 10.1371/journal.pone.0180786

Figure Lengend Snippet: (A) Flow cytometric analysis of cells treated with E1-Rluc8 and E1-EGFP. PC3 and HeLa cells (5 × 10 5 ) detached from culture plates were treated with E1-Rluc8 and E1-EGFP. The cells treated with E1-Rluc8 were stained with FITC-conjugated mouse anti-6×His antibody. Unstained cells or cells stained with FITC-conjugated secondary antibody (2 nd Ab) alone were used as controls. (B) Fluorescence microscopy images of the cells. PC3 and HeLa cells were stained with mouse anti-hEphA2 antibody and E1-Rluc8, and then detected with Alexa 555-conjugated anti-mouse IgG (red) and FITC-conjugated anti-6×His antibodies (green). Cell nuclei were stained with DAPI (blue). Scale bar, 10 μm. (C) Fluorescence microscopy images of xenograft tumor tissues. PC3 tumor tissues from transplanted nude mice were stained with the antibody combinations used in (B). Scale bar, 10 μm.

Article Snippet: Cells (1 × 10 4 ) were cultured on cover glasses on 6-well plates for 24 h. To detect bound E1-Rluc8, cells were incubated with 25.5 nM E1-Rluc8 and rabbit anti-hEphA2 antibody (1:10000 dilution, Santa Cruz Biotechnology), in 3 mL of culture medium containing 3% FBS at 37°C for 2 h. After the plates were washed with 1% BSA in PBS-T, they were incubated in FITC-conjugated anti-6×His (1:1000 dilution) and Alexa 555-conjugated anti-rabbit IgG antibodies (1:1000 dilution) for 2 h in the dark at room temperature (RT).

Techniques: Staining, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Engineering of monobody conjugates for human EphA2-specific optical imaging

doi: 10.1371/journal.pone.0180786

Figure Lengend Snippet: (A) Measurement of luminescence in mice injected with different concentrations of E1-Rluc8. PC3 and HeLa cells were transplanted into 6-week-old Balb/c nude mice via subcutaneous injection (n = 3). After tumor formation, the indicated amounts of E1-Rluc8 and coelenterazine were intravenously injected via the tail vein, and luminescence images were acquired at the indicated times with the NightOWL in vivo imaging system (left). The luminescence intensities measured in tumor areas at 6 h were graphed (right). (B) Luminescence maintenance in mice injected with E1-Rluc8 for 24 h. E1-Rluc8 (60 μg) and coelenterazine were intravenously injected via the tail vein into PC3 tumor mice (n = 5), and luminescence images were obtained at the indicated times (left). The luminescence intensities in tumor tissues were measured with the NightOWL in vivo imaging system and graphed (right). (C) Detection of the remaining E1-Rluc8 in PC3 tumor tissues. Tumor tissues were collected at the indicated times from mice injected with E1-Rluc8 and stained with FITC-conjugated anti-6×His antibodies (green). hEphA2 was stained with mouse anti-hEphA2 antibody and Alexa 555-conjugated anti-mouse IgG (red). Cell nuclei were stained with DAPI (blue). Scale bar, 10 μm.

Article Snippet: Cells (1 × 10 4 ) were cultured on cover glasses on 6-well plates for 24 h. To detect bound E1-Rluc8, cells were incubated with 25.5 nM E1-Rluc8 and rabbit anti-hEphA2 antibody (1:10000 dilution, Santa Cruz Biotechnology), in 3 mL of culture medium containing 3% FBS at 37°C for 2 h. After the plates were washed with 1% BSA in PBS-T, they were incubated in FITC-conjugated anti-6×His (1:1000 dilution) and Alexa 555-conjugated anti-rabbit IgG antibodies (1:1000 dilution) for 2 h in the dark at room temperature (RT).

Techniques: Injection, In Vivo Imaging, 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) 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: Cell pellets (1×10 6 cells) detached from cell culture plates were resuspended in SDS sample buffer and boiled for 5 min. After separation by 10% SDS-PAGE, proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA), and hEphA2 was detected with a mouse anti-hEphA2 antibody (1:500 dilution, Santa Cruz Biotechnology, CA) and horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000 dilution, Lifetechnology, Inc., Grand Island, NY) combination.

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: Cell pellets (1×10 6 cells) detached from cell culture plates were resuspended in SDS sample buffer and boiled for 5 min. After separation by 10% SDS-PAGE, proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA), and hEphA2 was detected with a mouse anti-hEphA2 antibody (1:500 dilution, Santa Cruz Biotechnology, CA) and horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000 dilution, Lifetechnology, Inc., Grand Island, NY) combination.

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: Cell pellets (1×10 6 cells) detached from cell culture plates were resuspended in SDS sample buffer and boiled for 5 min. After separation by 10% SDS-PAGE, proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA), and hEphA2 was detected with a mouse anti-hEphA2 antibody (1:500 dilution, Santa Cruz Biotechnology, CA) and horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000 dilution, Lifetechnology, Inc., Grand Island, NY) combination.

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: Cell pellets (1×10 6 cells) detached from cell culture plates were resuspended in SDS sample buffer and boiled for 5 min. After separation by 10% SDS-PAGE, proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA), and hEphA2 was detected with a mouse anti-hEphA2 antibody (1:500 dilution, Santa Cruz Biotechnology, CA) and horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000 dilution, Lifetechnology, Inc., Grand Island, NY) combination.

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: Cell pellets (1×10 6 cells) detached from cell culture plates were resuspended in SDS sample buffer and boiled for 5 min. After separation by 10% SDS-PAGE, proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA), and hEphA2 was detected with a mouse anti-hEphA2 antibody (1:500 dilution, Santa Cruz Biotechnology, CA) and horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000 dilution, Lifetechnology, Inc., Grand Island, NY) combination.

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

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: Cells (2.5×10 5 ) were stained with 100 μL of mouse anti-hEphA2 antibody (1:100 dilution, R&D Systems, MN) for 30 min on ice.

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: Cells (2.5×10 5 ) were stained with 100 μL of mouse anti-hEphA2 antibody (1:100 dilution, R&D Systems, MN) for 30 min on ice.

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: Cells (2.5×10 5 ) were stained with 100 μL of mouse anti-hEphA2 antibody (1:100 dilution, R&D Systems, MN) for 30 min on ice.

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: Cells (2.5×10 5 ) were stained with 100 μL of mouse anti-hEphA2 antibody (1:100 dilution, R&D Systems, MN) for 30 min on ice.

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: Cells (2.5×10 5 ) were stained with 100 μL of mouse anti-hEphA2 antibody (1:100 dilution, R&D Systems, MN) for 30 min on ice.

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