Recombinant human egf

Manufactured by BD

Sourced in United States

Recombinant human Epidermal Growth Factor (EGF) is a laboratory product that functions as a growth factor. It is a polypeptide that stimulates cell growth and differentiation.

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14 protocols using recombinant human egf

EGFR Signaling Pathway Protein Analysis

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Human recombinant EGF was from BD Bioscience. EGF-Rh, EGF-A647 and Trf-A647 were purchased from Invitrogen (Carlsbad, California, USA). PD158780 was purchased from Calbiochem. Other chemicals were from Sigma or Thermo Fisher Scientific unless indicated otherwise.
Monoclonal antibody to phosphotyrosines (PY20) conjugated to horseradish peroxidase (HRP), and monoclonal antibody to c-Cbl (#610442) were from BD Transduction Laboratories (San Diego, CA, USA); rabbit polyclonal antibodies to EGFR (1005), Grb2, Cbl-b (H-454) and epsin1 (H-130), and goat polyclonal antibody to epsin2 (C-16), NEDD4 (A-16) and UbcH5b/c (C-15) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); monoclonal antibody 528 to EGFR (mAb528) was from American Type Culture Collection (Manassas, VA, USA); monoclonal mouse antibody to EGFR phosphotyrosine 1068 (pY1068) was from Cell Signaling Technology (Beverly, MA, USA). Polyclonal rabbit antibodies to Eps15 (577) and Eps15R (860) were kindly provided by Dr. P. P. Di Fiore (European Institute of Oncology, Milan, Italy). Rabbit polyclonal antibody to CHC was from Abcam. Rabbit monoclonal antibody to Tom1L was from Epitomics (#0405). Rabbit polyclonal antibodies to FCHo1 and FCHo2 were a gift from Dr. Linton Traub (University of Pittsburgh). Rabbit polyclonal antibody to actin was from Sigma.

Fortian A., Dionne L.K., Hong S.H., Kim W., Gygi S.P., Watkins S, & Sorkin A. (2015). Endocytosis of ubiquitylation-deficient EGF receptor mutants via clathrin coated pits is mediated by ubiquitylation. Traffic (Copenhagen, Denmark), 16(11), 1137-1154.

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EGFR Signaling Pathway Activation Assay

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Human recombinant EGF was from BD Bioscience. EGF-Rh was from Invitrogen. Monoclonal antibody to EGFR phosphotyrosine 1068 (pY1068), polyclonal antibody to α-actinin, polyclonal antibody to ERK1/2 and monoclonal antibody to phosphorylated ERK1/2 were from Cell Signaling Technology (Beverly, MA). EGFR monoclonal antibody 04–105 were from Transduction Laboratories. PD158780 was from Calbiochem. MG-B-Tau and MG-Bis-SA were synthesized by the Bruchez lab (Perkins et al., 2018b (link); Yan et al., 2015 (link)). All chemicals were from ThermoFisher unless stated otherwise.

Larsen M.B., Perez Verdaguer M., Schmidt B.F., Bruchez M.P., Watkins S.C, & Sorkin A. (2019). Generation of endogenous pH-sensitive EGF receptor and its application in high-throughput screening for proteins involved in clathrin-mediated endocytosis. eLife, 8, e46135.

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Ovarian Cancer Cell Line Maintenance

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The SK-OV-3 and Caov-3 human ovarian cancer cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyClone; GE Healthcare Bio-Sciences, Logan, UT, USA), penicillin (100 U/ml) and streptomycin (100 µg/ml; GE Healthcare Bio-Sciences), and were cultured at 37°C in a 5% CO₂ atmosphere. Human recombinant EGF was purchased from BD Biosciences (San Jose, CA, USA). HA was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Sorafenib (BAY43-9006, Nexavar®) was purchased from LC Laboratories (Woburn, MA, USA).

Park G.B., Ko H.S, & Kim D. (2017). Sorafenib controls the epithelial-mesenchymal transition of ovarian cancer cells via EGF and the CD44-HA signaling pathway in a cell type-dependent manner. Molecular Medicine Reports, 16(2), 1826-1836.

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Cell Culture Conditions for Cancer Research

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The cell lines used were HF1 (gift from Mary Davis, University of Michigan), HPNE, Panc1, MiaPaCa2, BxPC3, UM16, UM28, UM59 and HEK293 (gifts from Diane Simeone University of Michigan), HeLa, GM12878 and K562 (ATCC) and LN428 (gift from Rob Sobol, University of Pittsburgh). The media used were MEM with 10% FBS and antibiotics (HF1), DMEM with 10% FBS and Pen/Strep (Panc1, MiaPaCa2, UM16, UM28, UM59, HEK293), RPMI1640 with 10% FBS and antibiotics (BxPC3), F12K with 10% FBS and Pen/Strep (HeLa), RPMI1640 with 15% FBS (GM12878), IMDM with 10% FBS (K562), and MEMalpha with 10% FBS, antibiotics, gentamycin and puromycin (LN428). The HPNE cells were grown in 75% DMEM without glucose (Sigma D-5030) containing 2 mM L-glutamine (Sigma G7513), 1.5g/L sodium bicarbonate (Sigma S 4019), 25% Medium M3 Base (Incell Corp, M300F-500) with the additives 10 ng/mL human recombinant EGF (BD Sciences 354052), 5.5 mM D-glucose (1g/L) (Sigma G8644) and 750 ng/mL puromycin dihydrochloride (Invitrogen A11138-02).

Lefkofsky H.B., Veloso A, & Ljungman M. (2014). Transcriptional and Post-Transcriptional Regulation of Nucleotide Excision Repair Genes in Human Cells. Mutation research, 776, 9-15.

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DMEM and FBS Cell Culture Protocol

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Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen (Carlsbad, CA). Dulbecco’s phosphate buffered saline (DPBS) was purchased from Mediatech Inc. (Herndon, VA). LB broth (BLF-7030) was from KD Medical (Columbia, MD). Sodium orthovanadate was from Fisher Chemicals (Fairlawn, NJ). NP40 Cell Lysis Buffer (FNN0021) was from ThermoFisher Scientific (Waltham, MA). Recombinant human EGF was purchased from BD Biosciences, Inc. (San Jose, CA). Tissue culture plasticware and other laboratory consumables were purchased from commercial sources.

Liyasova M.S., Ma K., Voeller D., Ryan P.E., Chen J., Klevit R.E, & Lipkowitz S. (2019). Cbl interacts with multiple E2s in vitro and in cells. PLoS ONE, 14(5), e0216967.

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Cell Culture Protocol for RET Signaling

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Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin sulfate were obtained from Invitrogen (Carlsbad, CA). Dulbecco’s phosphate buffered saline (D-PBS) was purchased from Mediatech Inc. (Herndon, VA). Tissue culture plastic ware and other laboratory consumables were purchased from commercial sources. Recombinant human EGF (#354052) was obtained from BD Biosciences (Bedford, MA). MG-132 (#474790) was purchased from Millipore (Billerica, MA), and cycloheximide (#C7698) was purchased from Sigma Aldrich (St. Louis, MO). Recombinant human RET ligand (GDNF; #714-GR-100) and exogenous co-receptor (GFRα1; #212-GD-010/CF) were purchased from R&D Systems (Minneapolis, MN).

Kales S.C., Nau M.M., Merchant A.S, & Lipkowitz S. (2014). Enigma Prevents Cbl-c-Mediated Ubiquitination and Degradation of RETMEN2A. PLoS ONE, 9(1), e87116.

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Establishment and Culture of Prostate Cell Line

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The PKV cell line was generated by FACS sorting CD45^-CD31^-Ter119^-EpCAM⁺GFP^- epithelial cells from the prostates of 10 week old CPKV mice and culturing them in 0.2% gelatin-coated 10 cm dishes with Dulbecco’s modified eagle medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) containing 1% Pen/Strep, 10% Fetal Bovine Serum (FBS) (Omega Scientific, Tarzana, CA, USA), 25 μg /mL bovine pituitary extract, 5 μg /mL insulin (Invitrogen, Grand Island, NY, USA), and 6 ng/mL recombinant human EGF (BD Biosciences, San Jose, CA, USA). LNCaP cells were grown in RPMI-1640 media (Sigma-Aldrich) with 1% Pen/Strep and 10% FBS. For studies carried out in the context of androgen deprivation, media containing 10% charcoal dextran-treated (CDT) FBS (BD Biosciences) was used.

Ruscetti M., Dadashian E.L., Guo W., Quach B., Mulholland D.J., Park J.W., Tran L.M., Kobayashi N., Bianchi-Frias D., Xing Y., Nelson P.S, & Wu H. (2015). HDAC Inhibition Impedes Epithelial-Mesenchymal Plasticity and Suppresses Metastatic, Castration-Resistant Prostate Cancer. Oncogene, 35(29), 3781-3795.

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Cell Culture and Treatment Protocol

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Dulbecco’s modified Eagle’s medium (DMEM), RPMI, and fetal bovine serum (FBS) were obtained from Invitrogen. Dulbecco’s phosphate buffered saline was purchased from Mediatech Inc. LB broth (BLF-7030) was from KD Medical. Sodium orthovanadate was from Fisher Chemicals. NP40 Cell Lysis Buffer (FNN0021), Geneticin Selective Antibiotic (G418 Sulfate, 10131027), and OptiMEM medium (31985062) were from ThermoFisher Scientific. Puromycin dihydrochloride (P9620) and leupeptin (L9783) were from Sigma Aldrich. Recombinant human EGF was purchased from BD Biosciences, Inc. Erlotinib (S1023), bortezomib (S1013), MG132 (S2619), chloroquine (S6999), cabozantinib (S1119), defactinib (S7654), and simvastatin (S1792) were from Selleckchem. Tissue culture plastic ware and other laboratory consumables were purchased from commercial sources.

Wisniewski D.J., Liyasova M.S., Korrapati S., Zhang X., Ratnayake S., Chen Q., Gilbert S.F., Catalano A., Voeller D., Meerzaman D., Guha U., Porat-Shliom N., Annunziata C.M, & Lipkowitz S. (2022). Flotillin-2 regulates epidermal growth factor receptor activation, degradation by Cbl-mediated ubiquitination, and cancer growth. The Journal of Biological Chemistry, 299(1), 102766.

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EGF Signaling Pathway Modulation

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Recombinant human EGF was from BD Biosciences. EGF-Rh was from Molecular Probes (Invitrogen). Mouse monoclonal antibody to EGFR phosphotyrosine 1068 (pY1068), phosphorylated ERK1/2, phosphorylated AKT, phosphorylated STAT3, MEK1/2 and c-Cbl; and rabbit polyclonal antibody to ERK1/2, phosphorylated MEK1/2, phosphorylated PLCγ1, phosphorylated SFKs, AKT and α-actinin were from Cell Signaling Technology (Danvers, MA). Polyclonal rabbit antibody to EGFR (1005) and mouse monoclonal to ubiquitin (P4D1) were from Santa Cruz Biotechnology (Dallas, TX). c-Cbl antibody was from BD Bioscience (610442) (San Jose, CA). EGFR-528 monoclonal antibody was from ATCC (Manassas, VA). EEA1 (ab2900) and Y1068 (ab32430) antibodies used for immunostaining of tumor cryosections were from AbCam (Cambridge, MA). Phosphotyrosine-specific antibody pY20 conjugated with horseradish peroxidase (pY20-HRP) was from BD Bioscience (San Jose, CA). Gefitinib was purchased from LC Laboratories (Woburn, MA), whereas Dyngo4a and Pitstop-2 were from AbCam (Cambridge, MA), Hoechst 33342 staining solution was from Thermofisher Scientific (Pittsburgh, PA)

Pinilla-Macua I., Grassart A., Duvvuri U., Watkins S.C, & Sorkin A. (2017). EGF receptor signaling, phosphorylation, ubiquitylation and endocytosis in tumors in vivo. eLife, 6, e31993.

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Biochemical Signaling Pathway Analysis

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Recombinant human EGF was purchased from BD Biosciences; EGF-Rh was from Molecular Probes (Invitrogen). Mouse monoclonal antibody to RAF1 was obtained from BD Transduction Laboratories (San Jose, CA). Rabbit monoclonal antibody to pSer338 RAF1, monoclonal antibody to MEK1/2, ERK1/2, polyclonal rabbit antibodies to phosphorylated MEK1/2, phosphorylated ERK1/2, and α-actinin were from Cell Signaling Technology (Danvers, MA). Secondary anti-mouse and anti-rabbit IRDye antibodies were from Li-COR. Sorafenib was purchased from Santa Cruz and stored as 10 mM stock solution in dimethyl sulfoxide (DMSO) at -20°C. CellMask, PCR, cloning, and other reagents and chemicals were from Thermo Fisher (Pittsburgh, PA).

Surve S.V., Myers P.J., Clayton S.A., Watkins S.C., Lazzara M.J, & Sorkin A. (2019). Localization dynamics of endogenous fluorescently labeled RAF1 in EGF-stimulated cells. Molecular Biology of the Cell, 30(4), 506-523.

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