Anti egfr antibody

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-EGFR antibody is a laboratory reagent used in scientific research. It is designed to specifically bind to and detect the epidermal growth factor receptor (EGFR) protein. The antibody can be used in various experimental techniques, such as Western blotting, immunohistochemistry, and immunoprecipitation, to study the expression and localization of EGFR in biological samples.

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52 protocols using anti egfr antibody

Immunohistochemical Analysis of EGFR in OSCC

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OSCC tumors with surrounding normal tissue were fixed in 10% formalin, embedded in paraffin, sectioned (10-μm thickness), and stained with hematoxylin and eosin (H&E) or processed for IHC. For IHC, 10-μm thick paraffin-embedded tissue sections were dewaxed in xylene and rehydrated in decreasing concentration of alcohol. Antigen retrieval was performed with EDTA (pH 9.0) at sub-boiling temperature for 15 minutes. Tissue sections were incubated in 1:50 dilution of anti-EGFR antibody (Cat# 4267, Cell Signaling Tech.) overnight at 4°C. Secondary antibody was applied for 30 minutes at 37°C and slides were developed with DAKO HRP-compatible DAB (Cat# SF-4100, Vector Laboratories, Burlingame, California, United States) and counterstained with Harris Hematoxylin. Images of H&E- and IHC-stained tissue sections were obtained using an inverted Keyence BZ-X810 microscope (Keyence, Itasca, Illinois, United States). A Plan Apo 10x, 0.45 NA air objective (Nikon, Tokyo, Japan) and a monochrome CCD (colorized with LC filter) were used to capture images. Histology images were graded by two experienced pathologists from Stanford and MGH.

Pal R., Hom M., van den Berg N.S., Lwin T.M., Lee Y.J., Prilutskiy A., Faquin W., Yang E., Saladi S.V., Varvares M.A., Rosenthal E.L, & Kumar A.T. (2022). First clinical results of fluorescence lifetime-enhanced tumor imaging using receptor targeted fluorescent probes. Clinical cancer research : an official journal of the American Association for Cancer Research, 28(11), 2373-2384.

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Protein Co-Immunoprecipitation Assay

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Cells were washed once with modified Dulbecco’s phosphate-buffered saline and lysed in buffer containing 0.025 M Tris, 0.15 M NaCl, 0.0001 M EDTA, 1% NP-40, and 5% glycerol (pH 7.4). Protein G/A agarose resin (Thermo Fisher Scientific, Waltham, MA) was incubated for 2 hours with rabbit monoclonal anti-MET antibody (Cell Signaling Technologies), anti-EGFR antibody (Cell Signaling Technologies) or rabbit IgG control (Invitrogen, Carlsbad, CA) as a negative control and cell lysate at 1 mg of protein was mixed with antibody-coupled resin (Cell Signaling Technologies) overnight at 4°C. Co-immunoprecipitation was performed using the Pierce Co-IP Kit (Thermo Fisher Scientific). Immunoprecipitates were washed, eluted, and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Western blot was performed as described above.

Kim S., Kim T.M., Kim D.W., Kim S., Kim M., Ahn Y.O., Keam B, & Heo D.S. (2018). Acquired Resistance of MET-Amplified Non-small Cell Lung Cancer Cells to the MET Inhibitor Capmatinib. Cancer Research and Treatment : Official Journal of Korean Cancer Association, 51(3), 951-962.

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Western Blot Antibody Incubation Protocol

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Anti-YAP antibody (#14074) and anti-EGFR antibody (#4267) were purchased from Cell Signaling Technology (Beverly MA, USA). The transferred membranes were subsequently incubated overnight (more than 16 hr) at 4 °C with the primary antibody (1:1000) and then the secondary antibody (1:3000) for 1 hr. Chemoluminescence detection was performed by using the Pierce ECL Western Blotting Substrate (Thermo Scientific).

Huang C., Chen Z., Yang C., Chen L., Lai C., Zhang Y., Yuan W, & Jeong J.H. (2020). Combinational inhibition of EGFR and YAP reverses 5-Fu resistance in colorectal cancer. Journal of Cancer, 11(18), 5432-5439.

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EGFR Phosphorylation Assay in A549 and SNU-5 Cells

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Approximately 10⁶ A549 or SNU-5 cells/well were grown overnight in six-well plates and incubated for 15 min with 30 μg/ml of antibody in the absence or presence of 40 ng/ml EGF. After cell lysis, Western blots determined EGFR phosphorylation status with phospho-EGFR (Tyr1068) antibody (Cell Signaling 2234) and total EGFR protein using an anti-EGFR antibody (Cell Signaling 2232).

Neijssen J., Cardoso R.M., Chevalier K.M., Wiegman L., Valerius T., Anderson G.M., Moores S.L., Schuurman J., Parren P.W., Strohl W.R, & Chiu M.L. (2021). Discovery of amivantamab (JNJ-61186372), a bispecific antibody targeting EGFR and MET. The Journal of Biological Chemistry, 296, 100641.

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Quantification of Breast Cancer Cells and EGFR-CAR NK Cells by Flow Cytometry

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The breast cancer cells and EGFR‐CAR NK cells were quantitated or isolated by flow cytometry using several fluorescence‐conjugated antibodies, following the manufacturer's instructions. The following reagents were used for this analysis: anti‐human CD3‐PE‐Cy7, anti‐human CD56‐PE, anti‐human CD69‐APC‐Cy7, mouse control PE, mouse control APC‐Cy7, mouse control PE‐Cy7, and Human TruStain FcX™ blocking solution purchased from BioLegend; anti‐EGFR antibody purchased from Cell Signal Technology; and goat anti‐rabbit IgG purchased from Abcam. The flow cytometric analyses were performed in a BD™ flow cytometer. The data were analyzed using FlowJo v10 software.

Liu Y., Zhou Y., Huang K., Fang X., Li Y., Wang F., An L., Chen Q., Zhang Y., Shi A., Yu S, & Zhang J. (2020). Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor. Cell Proliferation, 53(8), e12858.

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Western Blotting Protein Analysis Protocol

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For Western blotting, cells were lysed by lysis buffer (1% NP-40, 10% glycerol, 150 mM NaCl, 100 mM Sodium phosphate pH 7.2, 1 × EDTA-free protease inhibitor cocktail from Roche) and then incubated on ice for 15 min. Following incubation, the samples were spun at 13,000 g for 15 min. The supernatants were collected, and protein concentrations were determined by bicinchoninic acid protein assay kit. For each sample, 50 μg total lysate was separated on 4–12% NuPAGE (Invitrogen). After electrophoresis, the gels were blotted onto PVDF membranes (Millipore) and blocked with blocking buffer (5% BSA in TBS) for 1 h, and then incubated with anti-EGFR antibody (Cell signaling), anti-EGFR phosphosite-specific antibody (Cell signaling), anti-CDK1 antibody (Abnova) and anti-CDK1 (Abgent) phosphosite-specific antibody by 1:1,000 diluted in blocking buffer. After washing with TBST (0.05% Tween-20 in TBS), the membranes were incubated with peroxidase-conjugated second antibodies for developing the signal.

Tsai C.F., Wang Y.T., Yen H.Y., Tsou C.C., Ku W.C., Lin P.Y., Chen H.Y., Nesvizhskii A.I., Ishihama Y, & Chen Y.J. (2015). Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics. Nature Communications, 6, 6622.

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Protein Expression Analysis Protocol

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After isolation, content determination and electrophoresis, proteins were elettroblotted [46 (link)] and incubated with a polyclonal rabbit anti-CRBP-1, anti-Creb1, anti-CD44, anti-c-Jun, anti-Nox4, anti-p53, anti-RXRα, anti-RARα (Santa Cruz Biotechnology), anti-RARβ, (Abcam), anti-phosphorylated v-akt murine Jesi AN, Italy) and then sequenced using PyroMark Q24 thymoma viral oncogene homolog (pAkt Ser⁴⁷³), anti-AKT (pan), anti-phosphorylated extracellular-signal-regulated kinases (pErk1/2), anti-phosphorylated epidermal growth factor receptor (anti-EGFR Thr669), anti-EGFR antibody (Cell Signaling Technology, Danvers, MA, USA), anti-keratin 5 (clone H-40, Santa Cruz Biotechnology), mouse anti-vimentin (clone J144, Abcam), anti-keratin 14 (LL001, Santa Cruz Biotechnology) and anti-total tubulin antibody (Sigma-Aldrich). Revelation and densitometric blot analysis were performed in three independent experiments and Akt and EGFR activity expressed as phospho/total protein ratio [47 (link)].

Doldo E., Costanza G., Ferlosio A., Pompeo E., Agostinelli S., Bellezza G., Mazzaglia D., Giunta A., Sidoni A, & Orlandi A. (2015). High expression of cellular retinol binding protein-1 in lung adenocarcinoma is associated with poor prognosis. Genes & Cancer, 6(11-12), 490-502.

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RAB39B Subcellular Localization in Neuronal Cells

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PC12 cells were transfected with a wild type or mutant (p.G192R) myc-tagged RAB39B construct as described in the previous section and differentiated with nerve growth factor (NGF) for 4 days. The cells were fixed in 4 % paraformaldehyde and then incubated overnight at 4 °C with rabbit anti-myc and anti-chromogranin A antibodies. SK-N-BE(2)C cells were transfected with GFP-tagged wild type or mutant RAB39B constructs (pEGFP-N1 vector; Clontech Laboratories, Mountain View, CA) using Lipofectamine. After retinoic acid-induced differentiation for 4 days the cells were fixed in 4 % paraformaldehyde and incubated overnight at 4 °C with anti-epidermal growth factor receptor (anti-EGFR) antibody (a plasma membrane marker; Cell Signaling Technology, Danvers, MA) using methods described elsewhere [22 (link)]. Hoechst 33342 was used for counterstaining and confocal analysis was performed using an Olympus IX81 microscope.

Mata I.F., Jang Y., Kim C.H., Hanna D.S., Dorschner M.O., Samii A., Agarwal P., Roberts J.W., Klepitskaya O., Shprecher D.R., Chung K.A., Factor S.A., Espay A.J., Revilla F.J., Higgins D.S., Litvan I., Leverenz J.B., Yearout D., Inca-Martinez M., Martinez E., Thompson T.R., Cholerton B.A., Hu S.C., Edwards K.L., Kim K.S, & Zabetian C.P. (2015). The RAB39B p.G192R mutation causes X-linked dominant Parkinson’s disease. Molecular Neurodegeneration, 10, 50.

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Molecular Characterization of Patient-Derived Xenograft Models

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Formalin-fixed and paraffin-embedded tissue blocks of the 50 PDX models were prepared. Candidate targets, including EGFR, HER3, MET, and PD-L1, were stained via immunohistochemistry (IHC) using anti-EGFR antibody (#4267, Cell Signaling Technology, Danvers, MA, USA), anti-HER3 antibody (#2708, Cell Signaling Technology), anti-MET antibody (#790-4430, Ventana Medical Systems, Tucson, AZ, USA), and anti-PD-L1 antibody (#M4420, Spring Bioscience Corp., Pleasanton, CA, USA) according to the manufacturers’ instructions. IHC results were evaluated according to a previously published method [11 (link)–13 (link)]. Fluorescent in situ hybridization (FISH) was performed for MET and EGFR genes using the MET/CEN7 Dual Color Probe Kit (Zytovision, Bremerhafen, Germany) and EGFR/CEN7 Dual Color Probe (Zytovision) according to the manufacturer’s instructions. Gene amplifications were defined as the ratio of MET/CEN7 ≥ 2.2 and EGFR/CEN7 ≥ 2.2. All of the IHC and FISH results were reviewed and scored by two independent pathologists blinded to each other.

Chen Z., Huang W., Tian T., Zang W., Wang J., Liu Z., Li Z., Lai Y., Jiang Z., Gao J, & Shen L. (2018). Characterization and validation of potential therapeutic targets based on the molecular signature of patient-derived xenografts in gastric cancer. Journal of Hematology & Oncology, 11, 20.

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Oridonin Cytotoxicity and EGFR Modulation

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Oridonin (≥98%, HPLC) was purchased from mingwang biotechology (China). FBS, penicillin/streptomycin, DMEM medium, and trypsin kit were obtained from Gibco (USA). EGF was purchased from R&D (USA) and anti-EGFR antibody was obtained from Cell Signaling (USA). N-acetyl-l-cysteine (NAC), Annexin V-FITC/PI (Annexin V-Fluorescein Isothiocyanate/Propidium Iodide) apoptosis detection kit and DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) ROS assay kit were purchased from Beyotime Institute of Biotechnology. RIPA lysis buffer, Anti-EGFR IgG, anti-β-actin IgG, anti-rabbit IgG were from Cell Signaling (USA).

Pi J., Jin H., Jiang J., Yang F., Cai H., Yang P., Cai J, & Chen Z.W. (2017). Single molecule force spectroscopy for in-situ probing oridonin inhibited ROS-mediated EGF-EGFR interactions in living KYSE-150 cells. Pharmacological research, 119, 479-489.

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