Structured Review

Affibody egfr binding affibody molecule
Specificity of 89 Zr-DFO-ZEGFR:2377 uptake in A431 xenografts and <t>EGFR-expressing</t> organs in mice at 3 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of non-labelled <t>affibody</t> molecules.
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Images

1) Product Images from "PET imaging of epidermal growth factor receptor expression in tumours using 89Zr-labelled ZEGFR:2377 affibody molecules"

Article Title: PET imaging of epidermal growth factor receptor expression in tumours using 89Zr-labelled ZEGFR:2377 affibody molecules

Journal: International Journal of Oncology

doi: 10.3892/ijo.2016.3369

Specificity of 89 Zr-DFO-ZEGFR:2377 uptake in A431 xenografts and EGFR-expressing organs in mice at 3 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of non-labelled affibody molecules.
Figure Legend Snippet: Specificity of 89 Zr-DFO-ZEGFR:2377 uptake in A431 xenografts and EGFR-expressing organs in mice at 3 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of non-labelled affibody molecules.

Techniques Used: Expressing, Mouse Assay, Injection

2) Product Images from "Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors"

Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors

Journal: Bioconjugate Chemistry

doi: 10.1021/bc500525b

Modular capacity of probes for labeling EGFR on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with FAP–affibody fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.
Figure Legend Snippet: Modular capacity of probes for labeling EGFR on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with FAP–affibody fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.

Techniques Used: Labeling, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Incubation, Imaging

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.
Figure Legend 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.

Techniques Used: Binding Assay, Incubation, Flow Cytometry, Cytometry, Fluorescence, Competitive Binding Assay, Labeling, Serial Dilution, Activation Assay, Western Blot, Microscopy, Imaging

3) Product Images from "Geometry and expression enhance enrichment of functional yeast-displayed ligands via cell panning"

Article Title: Geometry and expression enhance enrichment of functional yeast-displayed ligands via cell panning

Journal: Biotechnology and bioengineering

doi: 10.1002/bit.26001

The effect of washing on enrichment ratio and recovery of yeast displaying fibronectin domain ligands panned on EGFR mid cells Yeast displaying E6.2.6′, E6.2.6′ N78S and WT′ (affinities indicated) mixed 1:1,000 with non-displaying yeast were panned against MDA-MB-231. The enrichment (A) and yield (B) of binding ligands is presented as the mean ± standard deviation of 3–9 replicates.
Figure Legend Snippet: The effect of washing on enrichment ratio and recovery of yeast displaying fibronectin domain ligands panned on EGFR mid cells Yeast displaying E6.2.6′, E6.2.6′ N78S and WT′ (affinities indicated) mixed 1:1,000 with non-displaying yeast were panned against MDA-MB-231. The enrichment (A) and yield (B) of binding ligands is presented as the mean ± standard deviation of 3–9 replicates.

Techniques Used: Multiple Displacement Amplification, Binding Assay, Standard Deviation

The effect of washing and incubation conditions on enrichment ratio and recovery of yeast displaying fibronectin domain ligands panned on EGFR high cells Yeast displaying E6.2.6′, E6.2.6′ N78S, and WT′ (affinities indicated) mixed 1:1,000 with non-displaying yeast were panned against EGFR-expressing MDA-MB-468. The enrichment and yield of binding ligands is presented as the mean ± standard deviation of 3–9 replicates. (A and B) Selections were performed under baseline conditions with the exception of varied number of washing steps. (C and D) Selections were performed with the indicated modulation of incubation conditions.
Figure Legend Snippet: The effect of washing and incubation conditions on enrichment ratio and recovery of yeast displaying fibronectin domain ligands panned on EGFR high cells Yeast displaying E6.2.6′, E6.2.6′ N78S, and WT′ (affinities indicated) mixed 1:1,000 with non-displaying yeast were panned against EGFR-expressing MDA-MB-468. The enrichment and yield of binding ligands is presented as the mean ± standard deviation of 3–9 replicates. (A and B) Selections were performed under baseline conditions with the exception of varied number of washing steps. (C and D) Selections were performed with the indicated modulation of incubation conditions.

Techniques Used: Incubation, Expressing, Multiple Displacement Amplification, Binding Assay, Standard Deviation

4) Product Images from "In Vivo Imaging of Xenograft Tumors Using an Epidermal Growth Factor Receptor-Specific Affibody Molecule Labeled with a Near-infrared Fluorophore 1"

Article Title: In Vivo Imaging of Xenograft Tumors Using an Epidermal Growth Factor Receptor-Specific Affibody Molecule Labeled with a Near-infrared Fluorophore 1

Journal: Neoplasia (New York, N.Y.)

doi:

Specific binding and uptake of IRDye800CW-labeled Affibody molecules. (A) The protein expression levels of EGFR and HER2 in MDA-MB-231 (MDA231), A431, SKOV3, and SKBR3 cells. Actin served as an internal control. The relative expression levels were calculated
Figure Legend Snippet: Specific binding and uptake of IRDye800CW-labeled Affibody molecules. (A) The protein expression levels of EGFR and HER2 in MDA-MB-231 (MDA231), A431, SKOV3, and SKBR3 cells. Actin served as an internal control. The relative expression levels were calculated

Techniques Used: Binding Assay, Labeling, Expressing, Multiple Displacement Amplification

The effect of EGF and EGFR-specific Affibody (Eaff) on EGFR-mediated phosphorylation of EGFR and ERK1/2 (P44/42 MAPK) proteins. A431 cells were treated with either Eaff or EGF. Two concentrations (5 and 20 nM) for both Eaff (Eaff5 and Eaff20) and EGF
Figure Legend Snippet: The effect of EGF and EGFR-specific Affibody (Eaff) on EGFR-mediated phosphorylation of EGFR and ERK1/2 (P44/42 MAPK) proteins. A431 cells were treated with either Eaff or EGF. Two concentrations (5 and 20 nM) for both Eaff (Eaff5 and Eaff20) and EGF

Techniques Used:

5) Product Images from "Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors"

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

Journal: Journal of the American Chemical Society

doi: 10.1021/jacs.8b07601

(A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of the affibody as indicated. All samples were treated with PNGase to degloycosylate and visualize EGFR extracellular domain (∼70 kDa) as a distinct band. Each sample contained both an affibody and EGFR fragment unless otherwise indicated. Note that only the N23BP mutant gave a photoproduct, which corresponded to a mass increase of 10 kDa. Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) N23BP and EGFR mixed as in (A) or with the equimolar concentration (260 nM) bovine serum albumin (BSA) as indicated and irradiated for the time listed. A band corresponding to the EGFR-affibody conjugate appears at 5, 15, and 30 min (red arrows). Ladder proteins are (top to bottom) 100, 75, and 50 kDa.
Figure Legend Snippet: (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of the affibody as indicated. All samples were treated with PNGase to degloycosylate and visualize EGFR extracellular domain (∼70 kDa) as a distinct band. Each sample contained both an affibody and EGFR fragment unless otherwise indicated. Note that only the N23BP mutant gave a photoproduct, which corresponded to a mass increase of 10 kDa. Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) N23BP and EGFR mixed as in (A) or with the equimolar concentration (260 nM) bovine serum albumin (BSA) as indicated and irradiated for the time listed. A band corresponding to the EGFR-affibody conjugate appears at 5, 15, and 30 min (red arrows). Ladder proteins are (top to bottom) 100, 75, and 50 kDa.

Techniques Used: Polyacrylamide Gel Electrophoresis, SDS Page, Mutagenesis, Concentration Assay, Irradiation

(A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.
Figure Legend Snippet: (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.

Techniques Used: Polyacrylamide Gel Electrophoresis, SDS Page, Irradiation, Produced, Labeling, Transfection, Fluorescence, Incubation

Left: Spheroids were treated with 1 μ M N23BP (top) or WT (bottom) affibody for a total of 4 h (left column) and 20 h (middle and right columns) and additionally irradiated with 365 nm light for 30 min after 3.5 h incubation (right column). These spheroids were sectioned at approximately the same depth and imaged for Rhodamine and GFP distribution. Micrographs show Rhodamine signal within each section normalized by the average GFP intensity. Scale bars are 200 μ m. Right: Five spheroids were grown and irradiated for 1 h with the indicated N23BP affibody concentration in growth media. Affibody containing media was removed, and spheroids were lysed and loaded onto a PAGE gel and probed for affibody conjugates using an anti-T7 antibody. High molecular weight bands around the expected molecular weight of EGFR were observed only when the spheroid-affibody mixtures were irradiated, indicating photo-cross-linking.
Figure Legend Snippet: Left: Spheroids were treated with 1 μ M N23BP (top) or WT (bottom) affibody for a total of 4 h (left column) and 20 h (middle and right columns) and additionally irradiated with 365 nm light for 30 min after 3.5 h incubation (right column). These spheroids were sectioned at approximately the same depth and imaged for Rhodamine and GFP distribution. Micrographs show Rhodamine signal within each section normalized by the average GFP intensity. Scale bars are 200 μ m. Right: Five spheroids were grown and irradiated for 1 h with the indicated N23BP affibody concentration in growth media. Affibody containing media was removed, and spheroids were lysed and loaded onto a PAGE gel and probed for affibody conjugates using an anti-T7 antibody. High molecular weight bands around the expected molecular weight of EGFR were observed only when the spheroid-affibody mixtures were irradiated, indicating photo-cross-linking.

Techniques Used: Irradiation, Incubation, Concentration Assay, Polyacrylamide Gel Electrophoresis, Molecular Weight

6) Product Images from "Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with 99mTc using a peptide-based cysteine-containing chelator"

Article Title: Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with 99mTc using a peptide-based cysteine-containing chelator

Journal: International Journal of Oncology

doi: 10.3892/ijo.2016.3721

Specificity of 99m Tc-ZEGFR:2377 uptake in A431 xenografts and EGFR-expressing organs in mice at 3 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of non-labeled anti-EGFR antibody cetuximab. The data are presented as average (n=4) and SD.
Figure Legend Snippet: Specificity of 99m Tc-ZEGFR:2377 uptake in A431 xenografts and EGFR-expressing organs in mice at 3 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of non-labeled anti-EGFR antibody cetuximab. The data are presented as average (n=4) and SD.

Techniques Used: Expressing, Mouse Assay, Injection, Labeling

Tumor-to-organ ratios of 99m Tc-ZEGFR:2377 in BALB/C nu/nu mice bearing EGFR-expressing A431 xenografts. The data are presented as average (n=4) and SD.
Figure Legend Snippet: Tumor-to-organ ratios of 99m Tc-ZEGFR:2377 in BALB/C nu/nu mice bearing EGFR-expressing A431 xenografts. The data are presented as average (n=4) and SD.

Techniques Used: Mouse Assay, Expressing

Imaging of EGFR-expressing A431 xenografts in BALB/C nu/nu mice using 99m Tc-ZEGFR:2377 at 3 and 24 h after injection.
Figure Legend Snippet: Imaging of EGFR-expressing A431 xenografts in BALB/C nu/nu mice using 99m Tc-ZEGFR:2377 at 3 and 24 h after injection.

Techniques Used: Imaging, Expressing, Mouse Assay, Injection

Cellular processing of 99m Tc-ZEGFR:2377 by EGFR-expressing MDA468 (A) and A431(B) cell lines. Cells were incubated with 10 nM 99m Tc-ZEGFR:2377. The data are presented as average (n=3) and SD. Error bars are not seen because they are smaller than point symbols.
Figure Legend Snippet: Cellular processing of 99m Tc-ZEGFR:2377 by EGFR-expressing MDA468 (A) and A431(B) cell lines. Cells were incubated with 10 nM 99m Tc-ZEGFR:2377. The data are presented as average (n=3) and SD. Error bars are not seen because they are smaller than point symbols.

Techniques Used: Expressing, Incubation

Biodistribution of 99m Tc-ZEGFR:2377 in BALB/C nu/nu mice bearing EGFR-expressing A431 xenografts at 3 and 24 h after injection. The data are presented as average (n=4) and SD.
Figure Legend Snippet: Biodistribution of 99m Tc-ZEGFR:2377 in BALB/C nu/nu mice bearing EGFR-expressing A431 xenografts at 3 and 24 h after injection. The data are presented as average (n=4) and SD.

Techniques Used: Mouse Assay, Expressing, Injection

In vitro specificity of 99m Tc-ZEGFR:2377 binding to three different EGFR-expressing cell lines. Cells were incubated with 10 nM 99m Tc-ZEGFR:2377. A large molar excess of non-labeled ZEGFR:2377 or cetuximab was used for blocking of receptors. The data are presented as average (n=3–6) and SD.
Figure Legend Snippet: In vitro specificity of 99m Tc-ZEGFR:2377 binding to three different EGFR-expressing cell lines. Cells were incubated with 10 nM 99m Tc-ZEGFR:2377. A large molar excess of non-labeled ZEGFR:2377 or cetuximab was used for blocking of receptors. The data are presented as average (n=3–6) and SD.

Techniques Used: In Vitro, Binding Assay, Expressing, Incubation, Labeling, Blocking Assay

7) Product Images from "Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors"

Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors

Journal: Bioconjugate Chemistry

doi: 10.1021/bc500525b

Modular capacity of probes for labeling EGFR on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with FAP–affibody fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.
Figure Legend Snippet: Modular capacity of probes for labeling EGFR on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with FAP–affibody fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.

Techniques Used: Labeling, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Incubation, Imaging

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.
Figure Legend 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.

Techniques Used: Binding Assay, Incubation, Flow Cytometry, Cytometry, Fluorescence, Competitive Binding Assay, Labeling, Serial Dilution, Activation Assay, Western Blot, Microscopy, Imaging

8) Product Images from "Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors"

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

Journal: Journal of the American Chemical Society

doi: 10.1021/jacs.8b07601

(A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.
Figure Legend Snippet: (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.

Techniques Used: Polyacrylamide Gel Electrophoresis, SDS Page, Irradiation, Produced, Labeling, Transfection, Fluorescence, Incubation

9) Product Images from "Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors"

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

Journal: Journal of the American Chemical Society

doi: 10.1021/jacs.8b07601

(A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of the affibody as indicated. All samples were treated with PNGase to degloycosylate and visualize EGFR extracellular domain (∼70 kDa) as a distinct band. Each sample contained both an affibody and EGFR fragment unless otherwise indicated. Note that only the N23BP mutant gave a photoproduct, which corresponded to a mass increase of 10 kDa. Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) N23BP and EGFR mixed as in (A) or with the equimolar concentration (260 nM) bovine serum albumin (BSA) as indicated and irradiated for the time listed. A band corresponding to the EGFR-affibody conjugate appears at 5, 15, and 30 min (red arrows). Ladder proteins are (top to bottom) 100, 75, and 50 kDa.
Figure Legend Snippet: (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of the affibody as indicated. All samples were treated with PNGase to degloycosylate and visualize EGFR extracellular domain (∼70 kDa) as a distinct band. Each sample contained both an affibody and EGFR fragment unless otherwise indicated. Note that only the N23BP mutant gave a photoproduct, which corresponded to a mass increase of 10 kDa. Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) N23BP and EGFR mixed as in (A) or with the equimolar concentration (260 nM) bovine serum albumin (BSA) as indicated and irradiated for the time listed. A band corresponding to the EGFR-affibody conjugate appears at 5, 15, and 30 min (red arrows). Ladder proteins are (top to bottom) 100, 75, and 50 kDa.

Techniques Used: Polyacrylamide Gel Electrophoresis, SDS Page, Mutagenesis, Concentration Assay, Irradiation

(A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.
Figure Legend Snippet: (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.

Techniques Used: Polyacrylamide Gel Electrophoresis, SDS Page, Irradiation, Produced, Labeling, Transfection, Fluorescence, Incubation

Left: Spheroids were treated with 1 μ M N23BP (top) or WT (bottom) affibody for a total of 4 h (left column) and 20 h (middle and right columns) and additionally irradiated with 365 nm light for 30 min after 3.5 h incubation (right column). These spheroids were sectioned at approximately the same depth and imaged for Rhodamine and GFP distribution. Micrographs show Rhodamine signal within each section normalized by the average GFP intensity. Scale bars are 200 μ m. Right: Five spheroids were grown and irradiated for 1 h with the indicated N23BP affibody concentration in growth media. Affibody containing media was removed, and spheroids were lysed and loaded onto a PAGE gel and probed for affibody conjugates using an anti-T7 antibody. High molecular weight bands around the expected molecular weight of EGFR were observed only when the spheroid-affibody mixtures were irradiated, indicating photo-cross-linking.
Figure Legend Snippet: Left: Spheroids were treated with 1 μ M N23BP (top) or WT (bottom) affibody for a total of 4 h (left column) and 20 h (middle and right columns) and additionally irradiated with 365 nm light for 30 min after 3.5 h incubation (right column). These spheroids were sectioned at approximately the same depth and imaged for Rhodamine and GFP distribution. Micrographs show Rhodamine signal within each section normalized by the average GFP intensity. Scale bars are 200 μ m. Right: Five spheroids were grown and irradiated for 1 h with the indicated N23BP affibody concentration in growth media. Affibody containing media was removed, and spheroids were lysed and loaded onto a PAGE gel and probed for affibody conjugates using an anti-T7 antibody. High molecular weight bands around the expected molecular weight of EGFR were observed only when the spheroid-affibody mixtures were irradiated, indicating photo-cross-linking.

Techniques Used: Irradiation, Incubation, Concentration Assay, Polyacrylamide Gel Electrophoresis, Molecular Weight

10) Product Images from "Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors"

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

Journal: Journal of the American Chemical Society

doi: 10.1021/jacs.8b07601

(A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of the affibody as indicated. All samples were treated with PNGase to degloycosylate and visualize EGFR extracellular domain (∼70 kDa) as a distinct band. Each sample contained both an affibody and EGFR fragment unless otherwise indicated. Note that only the N23BP mutant gave a photoproduct, which corresponded to a mass increase of 10 kDa. Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) N23BP and EGFR mixed as in (A) or with the equimolar concentration (260 nM) bovine serum albumin (BSA) as indicated and irradiated for the time listed. A band corresponding to the EGFR-affibody conjugate appears at 5, 15, and 30 min (red arrows). Ladder proteins are (top to bottom) 100, 75, and 50 kDa.
Figure Legend Snippet: (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of the affibody as indicated. All samples were treated with PNGase to degloycosylate and visualize EGFR extracellular domain (∼70 kDa) as a distinct band. Each sample contained both an affibody and EGFR fragment unless otherwise indicated. Note that only the N23BP mutant gave a photoproduct, which corresponded to a mass increase of 10 kDa. Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) N23BP and EGFR mixed as in (A) or with the equimolar concentration (260 nM) bovine serum albumin (BSA) as indicated and irradiated for the time listed. A band corresponding to the EGFR-affibody conjugate appears at 5, 15, and 30 min (red arrows). Ladder proteins are (top to bottom) 100, 75, and 50 kDa.

Techniques Used: Polyacrylamide Gel Electrophoresis, SDS Page, Mutagenesis, Concentration Assay, Irradiation

(A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.
Figure Legend Snippet: (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.

Techniques Used: Polyacrylamide Gel Electrophoresis, SDS Page, Irradiation, Produced, Labeling, Transfection, Fluorescence, Incubation

Left: Spheroids were treated with 1 μ M N23BP (top) or WT (bottom) affibody for a total of 4 h (left column) and 20 h (middle and right columns) and additionally irradiated with 365 nm light for 30 min after 3.5 h incubation (right column). These spheroids were sectioned at approximately the same depth and imaged for Rhodamine and GFP distribution. Micrographs show Rhodamine signal within each section normalized by the average GFP intensity. Scale bars are 200 μ m. Right: Five spheroids were grown and irradiated for 1 h with the indicated N23BP affibody concentration in growth media. Affibody containing media was removed, and spheroids were lysed and loaded onto a PAGE gel and probed for affibody conjugates using an anti-T7 antibody. High molecular weight bands around the expected molecular weight of EGFR were observed only when the spheroid-affibody mixtures were irradiated, indicating photo-cross-linking.
Figure Legend Snippet: Left: Spheroids were treated with 1 μ M N23BP (top) or WT (bottom) affibody for a total of 4 h (left column) and 20 h (middle and right columns) and additionally irradiated with 365 nm light for 30 min after 3.5 h incubation (right column). These spheroids were sectioned at approximately the same depth and imaged for Rhodamine and GFP distribution. Micrographs show Rhodamine signal within each section normalized by the average GFP intensity. Scale bars are 200 μ m. Right: Five spheroids were grown and irradiated for 1 h with the indicated N23BP affibody concentration in growth media. Affibody containing media was removed, and spheroids were lysed and loaded onto a PAGE gel and probed for affibody conjugates using an anti-T7 antibody. High molecular weight bands around the expected molecular weight of EGFR were observed only when the spheroid-affibody mixtures were irradiated, indicating photo-cross-linking.

Techniques Used: Irradiation, Incubation, Concentration Assay, Polyacrylamide Gel Electrophoresis, Molecular Weight

11) Product Images from "Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake"

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

Journal: EJNMMI Research

doi: 10.1186/s13550-016-0213-8

PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

Techniques Used: Positron Emission Tomography

PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

Techniques Used: Positron Emission Tomography

12) Product Images from "Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake"

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

Journal: EJNMMI Research

doi: 10.1186/s13550-016-0213-8

PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

Techniques Used: Positron Emission Tomography

PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

Techniques Used: Positron Emission Tomography

Related Articles

Clone Assay:

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: It is noteworthy, that the top clones from the combinatorial library selection and the synthetic consensus design had two and three mutations (D53Y/K58Y or K4P/K7P/K58Y), respectively, that are rare in natural homologs (0–2%). .. This EGFR-binding affibody proved to be amenable to all three modes of engineering charge.

Synthesized:

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: .. The coding sequence for the EGFR binding affibody designated as ZEGFR:1907 or the nonbinding parent Z domain was synthesized by Integrated DNA Technologies and ligated into the pET21b+ vector, attaching an N-terminal T7 epitope tag and C-terminal 6xHis tag. .. The ZEGFR:1907 construct was further mutated via the Q5 site directed mutagenesis kit (New England Biolabs) to substitute a single codon for cysteine at one of several locations within the affibody sequence.

Neutralization:

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: In fact, this mutant had higher yield, 3.2 ±1.9-fold (p=0.05) higher affinity, and comparable stability to parental EA68 despite the neutralization of six charged residues. .. This EGFR-binding affibody proved to be amenable to all three modes of engineering charge.

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: Herein, these three approaches – consensus design, combinatorial library screening, and synthetic consensus design – were used to introduce numerous variations of six simultaneous charged-to-neutral mutations within an EGFR-binding affibody. .. A mutant with 3.2 ±1.9-fold enhanced affinity (1.7 ±0.5 nM Kd ), comparable stability (68 °C Tm ), and improved yield (7.0 ±0.5 mg/L), despite neutralization of six charged residues, was engineered.

Cytometry:

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
Article Snippet: The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377 -ST and ZTaq:3638 -ST). .. The proteins were site-specifically labeled with DyLight488 for flow cytometry and ex vivo tissue analyses or with 11 C for in vivo PET studies.

Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors
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 . .. Flow cytometry assays and fluorescence microscopy demonstrated that these probes label endogenous EGFR on A431 cells without disruption of EGFR function, and low nanomolar surface K d values were observed with the double-ZEGFR:1907 constructs.

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: High throughput sorting of a combinatorial library – using the genotype-phenotype linkage of yeast display ( ) and flow cytometry – expands the number of mutants that can be efficiently evaluated albeit with potential limitations on the characterization of some phenotypes. .. Herein, these three approaches – consensus design, combinatorial library screening, and synthetic consensus design – were used to introduce numerous variations of six simultaneous charged-to-neutral mutations within an EGFR-binding affibody.

Construct:

Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors
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 . .. Flow cytometry assays and fluorescence microscopy demonstrated that these probes label endogenous EGFR on A431 cells without disruption of EGFR function, and low nanomolar surface K d values were observed with the double-ZEGFR:1907 constructs.

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
Article Snippet: DNA constructions and expression of Sel-tagged Affibody molecules The EGFR-binding Affibody molecule ZEGFR:2377 [ , ] and the irrelevant Taq polymerase-binding Affibody molecule ZTaq:3638 [ ] were fused with a C-terminal ST as previously described [ , ]. .. Gene segments encoding a hexahistidyl tag (H6 ), GFP, and a cleavage site for TEV-protease were introduced upstream of the Affibody molecules using polymerase chain reaction (PCR), to encode the constructs H6 -GFP-TEV-ZEGFR:2377 -ST and H6 -GFP-TEV-ZTaq:3638 -ST. Sequence-confirmed plasmids were transformed to BL21(DE3) cells (Novagen) already harboring the pSUABC plasmid [ ].

Article Title: Geometry and expression enhance enrichment of functional yeast-displayed ligands via cell panning
Article Snippet: EGFR-binding fibronectin clone E6.2.6′, affibody clone EA68, and Gp2 clone GαEGFR2.2.3 were tested. .. In the C-terminal fusion construct, all three scaffolds effectively enriched against highly expressing MDA-MB-468 ( ).

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: The coding sequence for the EGFR binding affibody designated as ZEGFR:1907 or the nonbinding parent Z domain was synthesized by Integrated DNA Technologies and ligated into the pET21b+ vector, attaching an N-terminal T7 epitope tag and C-terminal 6xHis tag. .. The ZEGFR:1907 construct was further mutated via the Q5 site directed mutagenesis kit (New England Biolabs) to substitute a single codon for cysteine at one of several locations within the affibody sequence.

Cell Surface Receptor Assay:

Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity
Article Snippet: The presence of various other receptors and proteins on the cell membrane should result in limited accessibility of the cell surface receptor by its ligand. .. Furthermore, the covalent labeling of targeting ligands with FITC for in vivo cellular studies may lead to the modification of some residues that are critical for targeting binding, consistent with the report by Lyakhovin the studies of interaction between EGFR-binding affibody and its receptor .

Incubation:

Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity
Article Snippet: .. Recently, it was reported by Frejd and co-workers that monomeric and dimeric EGFR-binding affibody (Z1907) were internalized as efficiently as EGFR monoclonal antibody cetuximab when they were incubated with A431 cells , . .. To investigate the internalization and subsequent sub-cellular location of heptameric ZEGFR ligand, the targeting ligand was incubated with A431 cells at 37°C for 2 h to promote its internalization.

Activity Assay:

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: Yet, consensus design is limited by the ability of nature to sample sequence space as well as the requisite retention of functional activity, which is different than our current activity of interest, i.e. , EGFR binding. .. Herein, these three approaches – consensus design, combinatorial library screening, and synthetic consensus design – were used to introduce numerous variations of six simultaneous charged-to-neutral mutations within an EGFR-binding affibody.

Expressing:

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
Article Snippet: .. DNA constructions and expression of Sel-tagged Affibody molecules The EGFR-binding Affibody molecule ZEGFR:2377 [ , ] and the irrelevant Taq polymerase-binding Affibody molecule ZTaq:3638 [ ] were fused with a C-terminal ST as previously described [ , ]. .. Gene segments encoding a hexahistidyl tag (H6 ), GFP, and a cleavage site for TEV-protease were introduced upstream of the Affibody molecules using polymerase chain reaction (PCR), to encode the constructs H6 -GFP-TEV-ZEGFR:2377 -ST and H6 -GFP-TEV-ZTaq:3638 -ST. Sequence-confirmed plasmids were transformed to BL21(DE3) cells (Novagen) already harboring the pSUABC plasmid [ ].

Article Title: Geometry and expression enhance enrichment of functional yeast-displayed ligands via cell panning
Article Snippet: EGFR-binding fibronectin clone E6.2.6′, affibody clone EA68, and Gp2 clone GαEGFR2.2.3 were tested. .. In the C-terminal fusion construct, all three scaffolds effectively enriched against highly expressing MDA-MB-468 ( ).

Modification:

Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity
Article Snippet: .. Furthermore, the covalent labeling of targeting ligands with FITC for in vivo cellular studies may lead to the modification of some residues that are critical for targeting binding, consistent with the report by Lyakhovin the studies of interaction between EGFR-binding affibody and its receptor . .. The receptor-bound heptameric ligands were efficiently internalized and further co-localized with the early endosome marker EEA1.

Transformation Assay:

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
Article Snippet: DNA constructions and expression of Sel-tagged Affibody molecules The EGFR-binding Affibody molecule ZEGFR:2377 [ , ] and the irrelevant Taq polymerase-binding Affibody molecule ZTaq:3638 [ ] were fused with a C-terminal ST as previously described [ , ]. .. Gene segments encoding a hexahistidyl tag (H6 ), GFP, and a cleavage site for TEV-protease were introduced upstream of the Affibody molecules using polymerase chain reaction (PCR), to encode the constructs H6 -GFP-TEV-ZEGFR:2377 -ST and H6 -GFP-TEV-ZTaq:3638 -ST. Sequence-confirmed plasmids were transformed to BL21(DE3) cells (Novagen) already harboring the pSUABC plasmid [ ].

Genetically Modified:

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: .. A previously developed EGFR-binding affibody known as ZEGFR1907 (referred to here as wildtype or WT) was genetically modified at its putative binding site. .. Because this affibody was developed from a nonbinding Z domain by mutating 13 amino acids clustered along two of the three alpha helices, amino acid positions within and surrounding this interface were chosen as sites for incorporating the photo-cross-linkable unit, benzophenone (BP).

Conjugation Assay:

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: In testing varying irradiation times, cross-linking between EGFR and N23BP affibody was observed to occur within 15 min. Affibodies containing BP at different amino acid sites may not have shown photo-cross-linking to EGFR because the BP group inhibited affibody-EGFR binding or the BP group was not properly oriented relative to EGFR once bound. .. The potential effect of amino acid mutation and BP conjugation on affibody affinity and overall stability was determined next.

Flow Cytometry:

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
Article Snippet: The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377 -ST and ZTaq:3638 -ST). .. The proteins were site-specifically labeled with DyLight488 for flow cytometry and ex vivo tissue analyses or with 11 C for in vivo PET studies.

Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors
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 . .. Flow cytometry assays and fluorescence microscopy demonstrated that these probes label endogenous EGFR on A431 cells without disruption of EGFR function, and low nanomolar surface K d values were observed with the double-ZEGFR:1907 constructs.

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: High throughput sorting of a combinatorial library – using the genotype-phenotype linkage of yeast display ( ) and flow cytometry – expands the number of mutants that can be efficiently evaluated albeit with potential limitations on the characterization of some phenotypes. .. Herein, these three approaches – consensus design, combinatorial library screening, and synthetic consensus design – were used to introduce numerous variations of six simultaneous charged-to-neutral mutations within an EGFR-binding affibody.

Ligation:

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
Article Snippet: The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377 -ST and ZTaq:3638 -ST). .. Changes in tracer uptake in A431 xenografts over time were also monitored, followed by ex vivo proximity ligation assays (PLA) of EGFR expressions.

Library Screening:

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: .. Herein, these three approaches – consensus design, combinatorial library screening, and synthetic consensus design – were used to introduce numerous variations of six simultaneous charged-to-neutral mutations within an EGFR-binding affibody. .. Mutant analysis at the clonal and population levels provides significant insight into the impact of charged-to-neutral mutations within this affibody domain.

Introduce:

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: A previously developed EGFR-binding affibody known as ZEGFR1907 (referred to here as wildtype or WT) was genetically modified at its putative binding site. .. As the WT affibodies do not possess cysteine residues, a cysteine was first inserted at specific amino acid sites, followed by reaction with maleimide-benzophenone to introduce a single BP per affibody.

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: .. Herein, these three approaches – consensus design, combinatorial library screening, and synthetic consensus design – were used to introduce numerous variations of six simultaneous charged-to-neutral mutations within an EGFR-binding affibody. .. Mutant analysis at the clonal and population levels provides significant insight into the impact of charged-to-neutral mutations within this affibody domain.

Labeling:

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
Article Snippet: The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377 -ST and ZTaq:3638 -ST). .. The proteins were site-specifically labeled with DyLight488 for flow cytometry and ex vivo tissue analyses or with 11 C for in vivo PET studies.

Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity
Article Snippet: Recently, it was reported by Frejd and co-workers that monomeric and dimeric EGFR-binding affibody (Z1907) were internalized as efficiently as EGFR monoclonal antibody cetuximab when they were incubated with A431 cells , . .. As illustrated in , bright punctuated dots can be observed using confocal microscopy, demonstrating that FITC labeled heptameric ZEGFR (green fluorescence) was internalized and co-localized (white arrows) with the early endosome marker EEA1 (red fluorescence).

Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors
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 . .. This is a recombinant and fluorogenic labeling reagent for native EGFR molecules.

Article Title: Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with 99mTc using a peptide-based cysteine-containing chelator
Article Snippet: To minimize the influence of such site, we introduced a cysteine challenge of the labeled conjugate before the final purification. .. The internalization by two EGFR-expressing cell lines was relatively slow , which is typical for EGFR-binding affibody molecules ( , ).

Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity
Article Snippet: .. Furthermore, the covalent labeling of targeting ligands with FITC for in vivo cellular studies may lead to the modification of some residues that are critical for targeting binding, consistent with the report by Lyakhovin the studies of interaction between EGFR-binding affibody and its receptor . .. The receptor-bound heptameric ligands were efficiently internalized and further co-localized with the early endosome marker EEA1.

Mutagenesis:

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: Neither increased irradiation time nor affibody concentration improved photo-cross-linking for N23BP or any other mutant. .. In testing varying irradiation times, cross-linking between EGFR and N23BP affibody was observed to occur within 15 min. Affibodies containing BP at different amino acid sites may not have shown photo-cross-linking to EGFR because the BP group inhibited affibody-EGFR binding or the BP group was not properly oriented relative to EGFR once bound.

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: A second mutant also had high affinity (1.8 ±0.4 nM), and the other two synthetic mutants had comparable affinities to the natural consensus series resulting in a set of mutants with significantly improved (p=0.05) affinities relative to natural consensus design ( ). .. This EGFR-binding affibody proved to be amenable to all three modes of engineering charge.

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: The coding sequence for the EGFR binding affibody designated as ZEGFR:1907 or the nonbinding parent Z domain was synthesized by Integrated DNA Technologies and ligated into the pET21b+ vector, attaching an N-terminal T7 epitope tag and C-terminal 6xHis tag. .. The ZEGFR:1907 construct was further mutated via the Q5 site directed mutagenesis kit (New England Biolabs) to substitute a single codon for cysteine at one of several locations within the affibody sequence.

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: Herein, these three approaches – consensus design, combinatorial library screening, and synthetic consensus design – were used to introduce numerous variations of six simultaneous charged-to-neutral mutations within an EGFR-binding affibody. .. Mutant analysis at the clonal and population levels provides significant insight into the impact of charged-to-neutral mutations within this affibody domain.

Imaging:

Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity
Article Snippet: Heptameric targeting ligands are internalized and present in the endosome One of exciting applications of targeting ligands is the delivery of imaging or therapeutic agents into specific cell types. .. Recently, it was reported by Frejd and co-workers that monomeric and dimeric EGFR-binding affibody (Z1907) were internalized as efficiently as EGFR monoclonal antibody cetuximab when they were incubated with A431 cells , .

Polymerase Chain Reaction:

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
Article Snippet: DNA constructions and expression of Sel-tagged Affibody molecules The EGFR-binding Affibody molecule ZEGFR:2377 [ , ] and the irrelevant Taq polymerase-binding Affibody molecule ZTaq:3638 [ ] were fused with a C-terminal ST as previously described [ , ]. .. Gene segments encoding a hexahistidyl tag (H6 ), GFP, and a cleavage site for TEV-protease were introduced upstream of the Affibody molecules using polymerase chain reaction (PCR), to encode the constructs H6 -GFP-TEV-ZEGFR:2377 -ST and H6 -GFP-TEV-ZTaq:3638 -ST. Sequence-confirmed plasmids were transformed to BL21(DE3) cells (Novagen) already harboring the pSUABC plasmid [ ].

Binding Assay:

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: .. In testing varying irradiation times, cross-linking between EGFR and N23BP affibody was observed to occur within 15 min. Affibodies containing BP at different amino acid sites may not have shown photo-cross-linking to EGFR because the BP group inhibited affibody-EGFR binding or the BP group was not properly oriented relative to EGFR once bound. .. BP is also capable of cross-linking to the affibody itself once it absorbs a photon, so it is possible that some mutants may have formed intramolecular cross-links rather than to EGFR.

Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity
Article Snippet: It is well known that the binding of EGF to EGFR promotes the internalization of the receptor through the endocytic pathway, and the internalized EGFR is strongly associated with an early endosome marker Early Endosome Antigen 1(EEA1) that is enriched in endosomes . .. Recently, it was reported by Frejd and co-workers that monomeric and dimeric EGFR-binding affibody (Z1907) were internalized as efficiently as EGFR monoclonal antibody cetuximab when they were incubated with A431 cells , .

Article Title: Influence of composition of cysteine-containing peptide-based chelators on biodistribution of 99mTc-labeled anti-EGFR affibody molecules
Article Snippet: Since internalization of affibody molecules after binding to EGFR is slow (Tolmachev et al. ; Garousi et al. , ), radiolabeled affibody molecules remain reversibly bound to the surface of hepatocytes and dissociate when blood concentration is decreased because of renal clearance. .. The second mechanism is independent on EGFR-binding, but depends on both charge and charge distribution in the C-terminus of affibody molecules (Garousi et al. ).

Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors
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 . .. This is a recombinant and fluorogenic labeling reagent for native EGFR molecules.

Article Title: In Vivo Imaging of Xenograft Tumors Using an Epidermal Growth Factor Receptor-Specific Affibody Molecule Labeled with a Near-infrared Fluorophore 1
Article Snippet: .. Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding Affibody molecule. ..

Article Title: Targeted PRINT Hydrogels: The Role of Nanoparticle Size and Ligand Density on Cell Association, Biodistribution, and Tumor Accumulation
Article Snippet: .. In this Letter, we varied targeting ligand density of an EGFR binding affibody on the surface of two different hydrogel PRINT nanoparticles (80 nm × 320 and 55 nm × 60 nm) and monitored effects on target-cell association, off-target phagocytic uptake, biodistribution, and tumor accumulation. .. Interestingly, variations in ligand density only significantly altered in vitro internalization rates for the 80 nm × 320 nm particle.

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: .. A previously developed EGFR-binding affibody known as ZEGFR1907 (referred to here as wildtype or WT) was genetically modified at its putative binding site. .. Because this affibody was developed from a nonbinding Z domain by mutating 13 amino acids clustered along two of the three alpha helices, amino acid positions within and surrounding this interface were chosen as sites for incorporating the photo-cross-linkable unit, benzophenone (BP).

Article Title: Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with 99mTc using a peptide-based cysteine-containing chelator
Article Snippet: The 99m Tc-ZEGFR:2377 retained capacity of specific binding to EGFR-expressing cells as it has been demonstrated by saturation of binding sites with both non-labeled ZEGFR:2377 and anti-EGFR antibody cetuximab ( ). .. The internalization by two EGFR-expressing cell lines was relatively slow , which is typical for EGFR-binding affibody molecules ( , ).

Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity
Article Snippet: .. Furthermore, the covalent labeling of targeting ligands with FITC for in vivo cellular studies may lead to the modification of some residues that are critical for targeting binding, consistent with the report by Lyakhovin the studies of interaction between EGFR-binding affibody and its receptor . .. The receptor-bound heptameric ligands were efficiently internalized and further co-localized with the early endosome marker EEA1.

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: .. The coding sequence for the EGFR binding affibody designated as ZEGFR:1907 or the nonbinding parent Z domain was synthesized by Integrated DNA Technologies and ligated into the pET21b+ vector, attaching an N-terminal T7 epitope tag and C-terminal 6xHis tag. .. The ZEGFR:1907 construct was further mutated via the Q5 site directed mutagenesis kit (New England Biolabs) to substitute a single codon for cysteine at one of several locations within the affibody sequence.

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: Yet, consensus design is limited by the ability of nature to sample sequence space as well as the requisite retention of functional activity, which is different than our current activity of interest, i.e. , EGFR binding. .. Herein, these three approaches – consensus design, combinatorial library screening, and synthetic consensus design – were used to introduce numerous variations of six simultaneous charged-to-neutral mutations within an EGFR-binding affibody.

Molecular Weight:

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: Successful photo-cross-linking of affibody to EGFR was determined by detecting the presence of a higher molecular weight band corresponding to the combined weight of both EGFR (70 kDa) extracellular domain and affibody (10 kDa) in the SDS-PAGE gel. .. In testing varying irradiation times, cross-linking between EGFR and N23BP affibody was observed to occur within 15 min. Affibodies containing BP at different amino acid sites may not have shown photo-cross-linking to EGFR because the BP group inhibited affibody-EGFR binding or the BP group was not properly oriented relative to EGFR once bound.

Marker:

Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity
Article Snippet: It is well known that the binding of EGF to EGFR promotes the internalization of the receptor through the endocytic pathway, and the internalized EGFR is strongly associated with an early endosome marker Early Endosome Antigen 1(EEA1) that is enriched in endosomes . .. Recently, it was reported by Frejd and co-workers that monomeric and dimeric EGFR-binding affibody (Z1907) were internalized as efficiently as EGFR monoclonal antibody cetuximab when they were incubated with A431 cells , .

Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity
Article Snippet: Furthermore, the covalent labeling of targeting ligands with FITC for in vivo cellular studies may lead to the modification of some residues that are critical for targeting binding, consistent with the report by Lyakhovin the studies of interaction between EGFR-binding affibody and its receptor . .. The receptor-bound heptameric ligands were efficiently internalized and further co-localized with the early endosome marker EEA1.

In Vivo:

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
Article Snippet: The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377 -ST and ZTaq:3638 -ST). .. The proteins were site-specifically labeled with DyLight488 for flow cytometry and ex vivo tissue analyses or with 11 C for in vivo PET studies.

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: In additional control tests, the N23BP affibody did not show cross-linking to other proteins such as bovine serum albumin , demonstrating both specificity for the intended cell receptor and its possible utility in vivo. .. In testing varying irradiation times, cross-linking between EGFR and N23BP affibody was observed to occur within 15 min. Affibodies containing BP at different amino acid sites may not have shown photo-cross-linking to EGFR because the BP group inhibited affibody-EGFR binding or the BP group was not properly oriented relative to EGFR once bound.

Article Title: Targeted PRINT Hydrogels: The Role of Nanoparticle Size and Ligand Density on Cell Association, Biodistribution, and Tumor Accumulation
Article Snippet: In this Letter, we varied targeting ligand density of an EGFR binding affibody on the surface of two different hydrogel PRINT nanoparticles (80 nm × 320 and 55 nm × 60 nm) and monitored effects on target-cell association, off-target phagocytic uptake, biodistribution, and tumor accumulation. .. However, in vivo , both particle sizes experienced significant changes in biodistribution and pharmacokinetics as a function of ligand density.

Article Title: Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with 99mTc using a peptide-based cysteine-containing chelator
Article Snippet: The internalization by two EGFR-expressing cell lines was relatively slow , which is typical for EGFR-binding affibody molecules ( , ). .. Blocking of EGFR receptors in A431 xenografts with the antibody cetuximab reduced significantly (P < 0.005) uptake of 99m Tc-ZEGFR:2377, which unambiguously demonstrated specificity of in vivo targeting ( ).

Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity
Article Snippet: .. Furthermore, the covalent labeling of targeting ligands with FITC for in vivo cellular studies may lead to the modification of some residues that are critical for targeting binding, consistent with the report by Lyakhovin the studies of interaction between EGFR-binding affibody and its receptor . .. The receptor-bound heptameric ligands were efficiently internalized and further co-localized with the early endosome marker EEA1.

Fluorescence:

Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity
Article Snippet: Recently, it was reported by Frejd and co-workers that monomeric and dimeric EGFR-binding affibody (Z1907) were internalized as efficiently as EGFR monoclonal antibody cetuximab when they were incubated with A431 cells , . .. As illustrated in , bright punctuated dots can be observed using confocal microscopy, demonstrating that FITC labeled heptameric ZEGFR (green fluorescence) was internalized and co-localized (white arrows) with the early endosome marker EEA1 (red fluorescence).

Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors
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 . .. This is a recombinant and fluorogenic labeling reagent for native EGFR molecules.

Multiple Displacement Amplification:

Article Title: Geometry and expression enhance enrichment of functional yeast-displayed ligands via cell panning
Article Snippet: EGFR-binding fibronectin clone E6.2.6′, affibody clone EA68, and Gp2 clone GαEGFR2.2.3 were tested. .. In the C-terminal fusion construct, all three scaffolds effectively enriched against highly expressing MDA-MB-468 ( ).

Isolation:

Article Title: Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with 99mTc using a peptide-based cysteine-containing chelator
Article Snippet: The overall isolated yield has decreased to 69±1% in this case, but the conjugate could withstand the cysteine challenge ( ). .. The internalization by two EGFR-expressing cell lines was relatively slow , which is typical for EGFR-binding affibody molecules ( , ).

Ex Vivo:

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
Article Snippet: The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377 -ST and ZTaq:3638 -ST). .. The proteins were site-specifically labeled with DyLight488 for flow cytometry and ex vivo tissue analyses or with 11 C for in vivo PET studies.

Purification:

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: In testing varying irradiation times, cross-linking between EGFR and N23BP affibody was observed to occur within 15 min. Affibodies containing BP at different amino acid sites may not have shown photo-cross-linking to EGFR because the BP group inhibited affibody-EGFR binding or the BP group was not properly oriented relative to EGFR once bound. .. First, microscale thermophoresis (NanoTemper) was used to measure the affinity of the N23BP affibody to purified EGFR and compared to WT affibody ( ).

Article Title: Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with 99mTc using a peptide-based cysteine-containing chelator
Article Snippet: To minimize the influence of such site, we introduced a cysteine challenge of the labeled conjugate before the final purification. .. The internalization by two EGFR-expressing cell lines was relatively slow , which is typical for EGFR-binding affibody molecules ( , ).

Sequencing:

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
Article Snippet: DNA constructions and expression of Sel-tagged Affibody molecules The EGFR-binding Affibody molecule ZEGFR:2377 [ , ] and the irrelevant Taq polymerase-binding Affibody molecule ZTaq:3638 [ ] were fused with a C-terminal ST as previously described [ , ]. .. Gene segments encoding a hexahistidyl tag (H6 ), GFP, and a cleavage site for TEV-protease were introduced upstream of the Affibody molecules using polymerase chain reaction (PCR), to encode the constructs H6 -GFP-TEV-ZEGFR:2377 -ST and H6 -GFP-TEV-ZTaq:3638 -ST. Sequence-confirmed plasmids were transformed to BL21(DE3) cells (Novagen) already harboring the pSUABC plasmid [ ].

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: This EGFR-binding affibody proved to be amenable to all three modes of engineering charge. .. This EGFR-binding affibody proved to be amenable to all three modes of engineering charge.

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: .. The coding sequence for the EGFR binding affibody designated as ZEGFR:1907 or the nonbinding parent Z domain was synthesized by Integrated DNA Technologies and ligated into the pET21b+ vector, attaching an N-terminal T7 epitope tag and C-terminal 6xHis tag. .. The ZEGFR:1907 construct was further mutated via the Q5 site directed mutagenesis kit (New England Biolabs) to substitute a single codon for cysteine at one of several locations within the affibody sequence.

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: Yet, consensus design is limited by the ability of nature to sample sequence space as well as the requisite retention of functional activity, which is different than our current activity of interest, i.e. , EGFR binding. .. Herein, these three approaches – consensus design, combinatorial library screening, and synthetic consensus design – were used to introduce numerous variations of six simultaneous charged-to-neutral mutations within an EGFR-binding affibody.

Positron Emission Tomography:

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
Article Snippet: The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377 -ST and ZTaq:3638 -ST). .. The proteins were site-specifically labeled with DyLight488 for flow cytometry and ex vivo tissue analyses or with 11 C for in vivo PET studies.

Blocking Assay:

Article Title: Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with 99mTc using a peptide-based cysteine-containing chelator
Article Snippet: The internalization by two EGFR-expressing cell lines was relatively slow , which is typical for EGFR-binding affibody molecules ( , ). .. Blocking of EGFR receptors in A431 xenografts with the antibody cetuximab reduced significantly (P < 0.005) uptake of 99m Tc-ZEGFR:2377, which unambiguously demonstrated specificity of in vivo targeting ( ).

Confocal Microscopy:

Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity
Article Snippet: Recently, it was reported by Frejd and co-workers that monomeric and dimeric EGFR-binding affibody (Z1907) were internalized as efficiently as EGFR monoclonal antibody cetuximab when they were incubated with A431 cells , . .. As illustrated in , bright punctuated dots can be observed using confocal microscopy, demonstrating that FITC labeled heptameric ZEGFR (green fluorescence) was internalized and co-localized (white arrows) with the early endosome marker EEA1 (red fluorescence).

Concentration Assay:

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: Neither increased irradiation time nor affibody concentration improved photo-cross-linking for N23BP or any other mutant. .. In testing varying irradiation times, cross-linking between EGFR and N23BP affibody was observed to occur within 15 min. Affibodies containing BP at different amino acid sites may not have shown photo-cross-linking to EGFR because the BP group inhibited affibody-EGFR binding or the BP group was not properly oriented relative to EGFR once bound.

Article Title: Influence of composition of cysteine-containing peptide-based chelators on biodistribution of 99mTc-labeled anti-EGFR affibody molecules
Article Snippet: Since internalization of affibody molecules after binding to EGFR is slow (Tolmachev et al. ; Garousi et al. , ), radiolabeled affibody molecules remain reversibly bound to the surface of hepatocytes and dissociate when blood concentration is decreased because of renal clearance. .. The second mechanism is independent on EGFR-binding, but depends on both charge and charge distribution in the C-terminus of affibody molecules (Garousi et al. ).

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
Article Snippet: DNA constructions and expression of Sel-tagged Affibody molecules The EGFR-binding Affibody molecule ZEGFR:2377 [ , ] and the irrelevant Taq polymerase-binding Affibody molecule ZTaq:3638 [ ] were fused with a C-terminal ST as previously described [ , ]. .. Gene expression was induced by adding isopropyl β-D-1-thiogalactopyranoside to a final concentration of 0.5 mM. l -Cysteine and selenite were added to the cultures to a final concentration of 1 μM and 5 nM, respectively, and cultivations were continued overnight at 25 °C.

Mouse Assay:

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
Article Snippet: The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377 -ST and ZTaq:3638 -ST). .. Kinetic scans with the 11 C-labeled proteins were performed in healthy mice and in mice bearing xenografts from human FaDu (squamous cell carcinoma) and A431 (epidermoid carcinoma) cell lines.

Chromatin Immunoprecipitation:

Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity
Article Snippet: First, the density and the accessibility of a receptor on the cell surface are quite different from that on the CM5 chip. .. Furthermore, the covalent labeling of targeting ligands with FITC for in vivo cellular studies may lead to the modification of some residues that are critical for targeting binding, consistent with the report by Lyakhovin the studies of interaction between EGFR-binding affibody and its receptor .

SDS Page:

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: Successful photo-cross-linking of affibody to EGFR was determined by detecting the presence of a higher molecular weight band corresponding to the combined weight of both EGFR (70 kDa) extracellular domain and affibody (10 kDa) in the SDS-PAGE gel. .. In testing varying irradiation times, cross-linking between EGFR and N23BP affibody was observed to occur within 15 min. Affibodies containing BP at different amino acid sites may not have shown photo-cross-linking to EGFR because the BP group inhibited affibody-EGFR binding or the BP group was not properly oriented relative to EGFR once bound.

Plasmid Preparation:

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
Article Snippet: DNA constructions and expression of Sel-tagged Affibody molecules The EGFR-binding Affibody molecule ZEGFR:2377 [ , ] and the irrelevant Taq polymerase-binding Affibody molecule ZTaq:3638 [ ] were fused with a C-terminal ST as previously described [ , ]. .. Gene segments encoding a hexahistidyl tag (H6 ), GFP, and a cleavage site for TEV-protease were introduced upstream of the Affibody molecules using polymerase chain reaction (PCR), to encode the constructs H6 -GFP-TEV-ZEGFR:2377 -ST and H6 -GFP-TEV-ZTaq:3638 -ST. Sequence-confirmed plasmids were transformed to BL21(DE3) cells (Novagen) already harboring the pSUABC plasmid [ ].

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: .. The coding sequence for the EGFR binding affibody designated as ZEGFR:1907 or the nonbinding parent Z domain was synthesized by Integrated DNA Technologies and ligated into the pET21b+ vector, attaching an N-terminal T7 epitope tag and C-terminal 6xHis tag. .. The ZEGFR:1907 construct was further mutated via the Q5 site directed mutagenesis kit (New England Biolabs) to substitute a single codon for cysteine at one of several locations within the affibody sequence.

Microscopy:

Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors
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 . .. Flow cytometry assays and fluorescence microscopy demonstrated that these probes label endogenous EGFR on A431 cells without disruption of EGFR function, and low nanomolar surface K d values were observed with the double-ZEGFR:1907 constructs.

Irradiation:

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: .. In testing varying irradiation times, cross-linking between EGFR and N23BP affibody was observed to occur within 15 min. Affibodies containing BP at different amino acid sites may not have shown photo-cross-linking to EGFR because the BP group inhibited affibody-EGFR binding or the BP group was not properly oriented relative to EGFR once bound. .. BP is also capable of cross-linking to the affibody itself once it absorbs a photon, so it is possible that some mutants may have formed intramolecular cross-links rather than to EGFR.

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: First, affibodies that can form covalent linkages to EGFR upon irradiation were produced by incorporating a photo-cross-linkable functional group at a specific amino acid site. .. A previously developed EGFR-binding affibody known as ZEGFR1907 (referred to here as wildtype or WT) was genetically modified at its putative binding site.

Functional Assay:

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: First, affibodies that can form covalent linkages to EGFR upon irradiation were produced by incorporating a photo-cross-linkable functional group at a specific amino acid site. .. A previously developed EGFR-binding affibody known as ZEGFR1907 (referred to here as wildtype or WT) was genetically modified at its putative binding site.

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: High throughput sorting can efficiently identify a population of functional sequences to serve as a synthetic basis for consensus design, which has been effectively implemented in evolving the chorismate mutase enzyme ( ). .. Herein, these three approaches – consensus design, combinatorial library screening, and synthetic consensus design – were used to introduce numerous variations of six simultaneous charged-to-neutral mutations within an EGFR-binding affibody.

Selection:

Article Title: In Vivo Imaging of Xenograft Tumors Using an Epidermal Growth Factor Receptor-Specific Affibody Molecule Labeled with a Near-infrared Fluorophore 1
Article Snippet: Friedman M, Nordberg E, Hoiden-Guthenberg I, Brismar H, Adams GP, Nilsson FY, Carlsson J, Stahl S. Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor. .. Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding Affibody molecule.

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: It is noteworthy, that the top clones from the combinatorial library selection and the synthetic consensus design had two and three mutations (D53Y/K58Y or K4P/K7P/K58Y), respectively, that are rare in natural homologs (0–2%). .. This EGFR-binding affibody proved to be amenable to all three modes of engineering charge.

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: Thus, considering the potential limitations of consensus design, we allowed homolog sequences to guide our selection of mutations while extending beyond natural consensus design. .. Herein, these three approaches – consensus design, combinatorial library screening, and synthetic consensus design – were used to introduce numerous variations of six simultaneous charged-to-neutral mutations within an EGFR-binding affibody.

In Vitro:

Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors
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 . .. In vitro fluorescence assays demonstrated that the binding of these dyes to the FAP–affibody fusions produced thousand-fold fluorescence enhancements, with high binding affinity and fast association rates.

Article Title: Targeted PRINT Hydrogels: The Role of Nanoparticle Size and Ligand Density on Cell Association, Biodistribution, and Tumor Accumulation
Article Snippet: In this Letter, we varied targeting ligand density of an EGFR binding affibody on the surface of two different hydrogel PRINT nanoparticles (80 nm × 320 and 55 nm × 60 nm) and monitored effects on target-cell association, off-target phagocytic uptake, biodistribution, and tumor accumulation. .. Interestingly, variations in ligand density only significantly altered in vitro internalization rates for the 80 nm × 320 nm particle.

Article Title: Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity
Article Snippet: The discrepancy of binding strength between in vitro and in vivo analyses is typical as observed in many other targeting ligands. .. Furthermore, the covalent labeling of targeting ligands with FITC for in vivo cellular studies may lead to the modification of some residues that are critical for targeting binding, consistent with the report by Lyakhovin the studies of interaction between EGFR-binding affibody and its receptor .

Proximity Ligation Assay:

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake
Article Snippet: The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377 -ST and ZTaq:3638 -ST). .. Changes in tracer uptake in A431 xenografts over time were also monitored, followed by ex vivo proximity ligation assays (PLA) of EGFR expressions.

Live Cell Imaging:

Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors
Article Snippet: Fluorescence is essential for dynamic live cell imaging, and affinity reagents are required for quantification of endogenous proteins. .. 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 .

Microscale Thermophoresis:

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: In testing varying irradiation times, cross-linking between EGFR and N23BP affibody was observed to occur within 15 min. Affibodies containing BP at different amino acid sites may not have shown photo-cross-linking to EGFR because the BP group inhibited affibody-EGFR binding or the BP group was not properly oriented relative to EGFR once bound. .. First, microscale thermophoresis (NanoTemper) was used to measure the affinity of the N23BP affibody to purified EGFR and compared to WT affibody ( ).

Produced:

Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors
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 . .. In vitro fluorescence assays demonstrated that the binding of these dyes to the FAP–affibody fusions produced thousand-fold fluorescence enhancements, with high binding affinity and fast association rates.

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: First, affibodies that can form covalent linkages to EGFR upon irradiation were produced by incorporating a photo-cross-linkable functional group at a specific amino acid site. .. A previously developed EGFR-binding affibody known as ZEGFR1907 (referred to here as wildtype or WT) was genetically modified at its putative binding site.

Activation Assay:

Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors
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 . .. This is a recombinant and fluorogenic labeling reagent for native EGFR molecules.

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors
Article Snippet: A previously developed EGFR-binding affibody known as ZEGFR1907 (referred to here as wildtype or WT) was genetically modified at its putative binding site. .. Benzophenone was chosen because it has been previously utilized as a photo-cross-linking agent for proteins upon activation with 365 nm light at intensities that do not significantly damage biological samples.

High Throughput Screening Assay:

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: High throughput sorting can efficiently identify a population of functional sequences to serve as a synthetic basis for consensus design, which has been effectively implemented in evolving the chorismate mutase enzyme ( ). .. Herein, these three approaches – consensus design, combinatorial library screening, and synthetic consensus design – were used to introduce numerous variations of six simultaneous charged-to-neutral mutations within an EGFR-binding affibody.

Recombinant:

Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors
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 . .. This is a recombinant and fluorogenic labeling reagent for native EGFR molecules.

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: This ‘synthetic consensus design’ identified a mutant – from only four tested – with 6 °C higher thermal stability, 6.4 ±4.3-fold stronger affinity, and 1.8 ±0.5-fold higher recombinant yield than EA35. .. This EGFR-binding affibody proved to be amenable to all three modes of engineering charge.

Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
Article Snippet: Herein, these three approaches – consensus design, combinatorial library screening, and synthetic consensus design – were used to introduce numerous variations of six simultaneous charged-to-neutral mutations within an EGFR-binding affibody. .. While all three approaches yielded a range of affinities, stabilities, and recombinant yields including several highly functional mutants, synthetic consensus design was the most effective.

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    Affibody egfr binding affibody molecule
    Specificity of 89 Zr-DFO-ZEGFR:2377 uptake in A431 xenografts and <t>EGFR-expressing</t> organs in mice at 3 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of non-labelled <t>affibody</t> molecules.
    Egfr Binding Affibody Molecule, supplied by Affibody, used in various techniques. Bioz Stars score: 87/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 87 stars, based on 3 article reviews
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    egfr binding affibody molecule - by Bioz Stars, 2020-02
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    83
    Affibody compact egfr binding affibody zegfr
    Modular capacity of probes for labeling <t>EGFR</t> on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with <t>FAP–affibody</t> fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.
    Compact Egfr Binding Affibody Zegfr, supplied by Affibody, used in various techniques. Bioz Stars score: 83/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    compact egfr binding affibody zegfr - by Bioz Stars, 2020-02
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    78
    Affibody egfr binding fibronectin
    The effect of washing on enrichment ratio and recovery of yeast displaying <t>fibronectin</t> domain ligands panned on <t>EGFR</t> mid cells Yeast displaying E6.2.6′, E6.2.6′ N78S and WT′ (affinities indicated) mixed 1:1,000 with non-displaying yeast were panned against MDA-MB-231. The enrichment (A) and yield (B) of binding ligands is presented as the mean ± standard deviation of 3–9 replicates.
    Egfr Binding Fibronectin, supplied by Affibody, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Specificity of 89 Zr-DFO-ZEGFR:2377 uptake in A431 xenografts and EGFR-expressing organs in mice at 3 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of non-labelled affibody molecules.

    Journal: International Journal of Oncology

    Article Title: PET imaging of epidermal growth factor receptor expression in tumours using 89Zr-labelled ZEGFR:2377 affibody molecules

    doi: 10.3892/ijo.2016.3369

    Figure Lengend Snippet: Specificity of 89 Zr-DFO-ZEGFR:2377 uptake in A431 xenografts and EGFR-expressing organs in mice at 3 h after injection. In the blocked group, receptors were saturated by pre-injection of large excess of non-labelled affibody molecules.

    Article Snippet: In the present study, an EGFR-binding affibody molecule (ZEGFR:2377) was site-specifically conjugated with a deferoxamine (DFO) chelator and labelled under mild conditions (room temperature and neutral pH) with a positron-emitting radionuclide 89 Zr.

    Techniques: Expressing, Mouse Assay, Injection

    Specific binding and uptake of IRDye800CW-labeled Affibody molecules. (A) The protein expression levels of EGFR and HER2 in MDA-MB-231 (MDA231), A431, SKOV3, and SKBR3 cells. Actin served as an internal control. The relative expression levels were calculated

    Journal: Neoplasia (New York, N.Y.)

    Article Title: In Vivo Imaging of Xenograft Tumors Using an Epidermal Growth Factor Receptor-Specific Affibody Molecule Labeled with a Near-infrared Fluorophore 1

    doi:

    Figure Lengend Snippet: Specific binding and uptake of IRDye800CW-labeled Affibody molecules. (A) The protein expression levels of EGFR and HER2 in MDA-MB-231 (MDA231), A431, SKOV3, and SKBR3 cells. Actin served as an internal control. The relative expression levels were calculated

    Article Snippet: Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding Affibody molecule.

    Techniques: Binding Assay, Labeling, Expressing, Multiple Displacement Amplification

    The effect of EGF and EGFR-specific Affibody (Eaff) on EGFR-mediated phosphorylation of EGFR and ERK1/2 (P44/42 MAPK) proteins. A431 cells were treated with either Eaff or EGF. Two concentrations (5 and 20 nM) for both Eaff (Eaff5 and Eaff20) and EGF

    Journal: Neoplasia (New York, N.Y.)

    Article Title: In Vivo Imaging of Xenograft Tumors Using an Epidermal Growth Factor Receptor-Specific Affibody Molecule Labeled with a Near-infrared Fluorophore 1

    doi:

    Figure Lengend Snippet: The effect of EGF and EGFR-specific Affibody (Eaff) on EGFR-mediated phosphorylation of EGFR and ERK1/2 (P44/42 MAPK) proteins. A431 cells were treated with either Eaff or EGF. Two concentrations (5 and 20 nM) for both Eaff (Eaff5 and Eaff20) and EGF

    Article Snippet: Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding Affibody molecule.

    Techniques:

    Modular capacity of probes for labeling EGFR on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with FAP–affibody fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. Scale bar 20 μm.

    Journal: Bioconjugate Chemistry

    Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors

    doi: 10.1021/bc500525b

    Figure Lengend Snippet: Modular capacity of probes for labeling EGFR on the cell surface. (A) Structures of the fluorogens used. The synthetic and analytical details were shown in Supporting Information or described previously. 26 A431 cell labeled with FAP–affibody fusions and various malachite green derivatives were analyzed by flow cytometry (B) and live-cell fluorescence microscopy (C). 5 × 10 5 /mL of cells were labeled with 250 nM of AFA or F followed by incubation with 100 nM of fluorogens for 5 min. Cells were then either analyzed by flow cytometry for mean fluorescence measurement or cell imaging. 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: Labeling, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Incubation, Imaging

    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.

    Journal: Bioconjugate Chemistry

    Article Title: Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors

    doi: 10.1021/bc500525b

    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

    The effect of washing on enrichment ratio and recovery of yeast displaying fibronectin domain ligands panned on EGFR mid cells Yeast displaying E6.2.6′, E6.2.6′ N78S and WT′ (affinities indicated) mixed 1:1,000 with non-displaying yeast were panned against MDA-MB-231. The enrichment (A) and yield (B) of binding ligands is presented as the mean ± standard deviation of 3–9 replicates.

    Journal: Biotechnology and bioengineering

    Article Title: Geometry and expression enhance enrichment of functional yeast-displayed ligands via cell panning

    doi: 10.1002/bit.26001

    Figure Lengend Snippet: The effect of washing on enrichment ratio and recovery of yeast displaying fibronectin domain ligands panned on EGFR mid cells Yeast displaying E6.2.6′, E6.2.6′ N78S and WT′ (affinities indicated) mixed 1:1,000 with non-displaying yeast were panned against MDA-MB-231. The enrichment (A) and yield (B) of binding ligands is presented as the mean ± standard deviation of 3–9 replicates.

    Article Snippet: EGFR-binding fibronectin clone E6.2.6′, affibody clone EA68, and Gp2 clone GαEGFR2.2.3 were tested.

    Techniques: Multiple Displacement Amplification, Binding Assay, Standard Deviation

    The effect of washing and incubation conditions on enrichment ratio and recovery of yeast displaying fibronectin domain ligands panned on EGFR high cells Yeast displaying E6.2.6′, E6.2.6′ N78S, and WT′ (affinities indicated) mixed 1:1,000 with non-displaying yeast were panned against EGFR-expressing MDA-MB-468. The enrichment and yield of binding ligands is presented as the mean ± standard deviation of 3–9 replicates. (A and B) Selections were performed under baseline conditions with the exception of varied number of washing steps. (C and D) Selections were performed with the indicated modulation of incubation conditions.

    Journal: Biotechnology and bioengineering

    Article Title: Geometry and expression enhance enrichment of functional yeast-displayed ligands via cell panning

    doi: 10.1002/bit.26001

    Figure Lengend Snippet: The effect of washing and incubation conditions on enrichment ratio and recovery of yeast displaying fibronectin domain ligands panned on EGFR high cells Yeast displaying E6.2.6′, E6.2.6′ N78S, and WT′ (affinities indicated) mixed 1:1,000 with non-displaying yeast were panned against EGFR-expressing MDA-MB-468. The enrichment and yield of binding ligands is presented as the mean ± standard deviation of 3–9 replicates. (A and B) Selections were performed under baseline conditions with the exception of varied number of washing steps. (C and D) Selections were performed with the indicated modulation of incubation conditions.

    Article Snippet: EGFR-binding fibronectin clone E6.2.6′, affibody clone EA68, and Gp2 clone GαEGFR2.2.3 were tested.

    Techniques: Incubation, Expressing, Multiple Displacement Amplification, Binding Assay, Standard Deviation