Egf recombinant human protein

Manufactured by Thermo Fisher Scientific

Sourced in United States

EGF recombinant human protein is a laboratory reagent produced by Thermo Fisher Scientific. It is a purified form of the epidermal growth factor (EGF) protein, which is a signaling molecule involved in cellular growth and differentiation. The core function of this product is to serve as a tool for researchers studying cellular processes related to EGF signaling in various experimental systems.

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17 protocols using egf recombinant human protein

Culturing Diverse Melanoma and Breast Cancer Cell Lines

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SK-MEL-28, WM164, WM793, and WM266-4 cell lines were a gift from Peter Leedman, and they were cultured in RPMI-1640 medium without phenol red (produced by the Harry Perkins Institute of Medical Research, Perth, Australia; formulated to ATCC specifications), supplemented with 10% heat-inactivated HyClone fetal bovine serum (FBS; GE Healthcare Life Sciences, Chicago, IL) and 1% antibiotic-antimycotic (Anti-Anti; Gibco, Fisher Scientific, Hampton, NH). Normal human melanocytes were a gift from Mel Ziman, and they were cultured in melanocyte growth medium M2 (PromoCell, Heidelberg, Germany). SUM159 cells were cultured in F-12 Nutrient Mixture (Gibco) supplemented with 5% FBS, 0.6% 1 M HEPES (Gibco), 5 μg/mL insulin, human recombinant, zinc solution (Gibco), 1 μg/mL hydrocortisone (Merck, Kenilworth, NJ), and 1% Anti-Anti. MCF-10A cells were cultured in DMEM/F-12 medium (Gibco), supplemented with 5% Normal Horse Serum (Life Technologies, Carlsbad, CA), 20 ng/mL EGF Recombinant Human Protein (Gibco), 0.01 mg/mL insulin, human recombinant, zinc solution, 500 ng/mL hydrocortisone, 100 ng/mL cholera toxin (Sigma-Aldrich, St. Louis, MO), and 1% Anti-Anti. BT-549, ZR-75-1, and MDA-MB-468 cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% Anti-Anti.

Moses C., Nugent F., Waryah C.B., Garcia-Bloj B., Harvey A.R, & Blancafort P. (2018). Activating PTEN Tumor Suppressor Expression with the CRISPR/dCas9 System. Molecular Therapy. Nucleic Acids, 14, 287-300.

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Investigating EGF Signaling Dynamics

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shCT, shHrs, and shTsg101 cells were preincubated for 1 h in growth medium in presence of 10 μg/mL of cycloheximide. Cells were then cultured in growth medium in presence of cycloheximide (10 μg/mL) and with EGF Recombinant Human protein (50 ng/mL; Gibco) for 15, 60, and 120 min. After these time points, cells were recovered and analyzed by Western blotting.

Coudert L., Osseni A., Gangloff Y.G., Schaeffer L, & Leblanc P. (2021). The ESCRT-0 subcomplex component Hrs/Hgs is a master regulator of myogenesis via modulation of signaling and degradation pathways. BMC Biology, 19, 153.

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Synthesis and Characterization of Nanoparticles

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Gold (III) chloride trihydrate, Silver Nitrate, L-ascorbic Acid, Hyaluronic Acid, Rosmarinic Acid (RA), Oleic Acid, Holo-Transferrin human (powder, grade 97%), and Lapatinib (98%) were acquired from Sigma-Aldrich (St. Louis, MO, USA). EGF Recombinant Human Protein was supplied from Gibco (ThermoFisher Scientific, Waltham, MA, USA). Bradford Dye Reagent was purchased from Alfa Aesar (ThermoFisher Scientific, Waltham, MA, USA). Water MilliQ by Merck Millipore (Burlington, MA, USA). All chemical products and solvents used were of analytical purity grade.

Amaral M., Charmier A.J., Afonso R.A., Catarino J., Faísca P., Carvalho L., Ascensão L., Coelho J.M., Gaspar M.M, & Reis C.P. (2021). Gold-Based Nanoplataform for the Treatment of Anaplastic Thyroid Carcinoma: A Step Forward. Cancers, 13(6), 1242.

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Cell Line Characterization and Treatment

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Cell lines were obtained, authenticated, and cultured as recommended by the American Type Culture Collection (ATCC). All cell lines were tested and negative for mycoplasma. H1975 (EGFR^{L858R, T790M}), A549 (KRAS^G12S), HCC364 (BRAF^V600E), H3122 (EML4-ALK variant 1), and AGS cells were obtained from ATCC. H1975 10-1 and H1975 1-1 metastatic derivatives were obtained from Clovis Oncology and renamed H1975 M1 and H1975 M2, respectively, both were engineered as previously described². All cell lines were maintained at 37 °C in a humidified atmosphere at 5% CO₂ and grown RPMI 1640 media supplemented with 10% FBS, 100 IU/ml penicillin and 100ug/ml streptomycin.
Rociletinib was obtained from Clovis. EGF recombinant human protein was obtained from ThermoFisher.

Okimoto R.A., Breitenbuecher F., Olivas V.R., Wu W., Gini B., Hofree M., Asthana S., Hrustanovic G., Flanagan J., Tulpule A., Blakely C.M., Haringsma H.J., Simmons A.D., Gowen K., Suh J., Miller V.A., Ali S., Schuler M, & Bivona T.G. (2016). Inactivation of Capicua drives cancer metastasis. Nature genetics, 49(1), 87-96.

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Neural Cell Culture Protocol

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Dulbecco’s modified Eagle Medium (DMEM), neural basal medium, B27 supplement, KnockOut™ DMEM/F-12 medium, StemPro™ Neural Supplement, StemPro Accutase, FGF-basic (AA 10–155) recombinant human Protein, EGF Recombinant Human Protein were purchased from ThermoFisher Canada. Modified ITS culture supplement was purchased from Wisent Canada., Poly-D-lysin and laminin were obtained from Sigma-Aldrich (St.Louis, MO, USA). The antibodies used were as follows: Antibodies: Anti-MAP2 antibody: Abcam Cat# ab28032; Anti-MeCP2 antibody: Abcam Cat# ab2828; Anti-PDGFR-alpha: Abcam Cat# ab96569; Anti-Myelin Basic Protein antibody: Abcam Cat# ab218011; Anti-GFAP Antibody: ThermoFisher Scientific Cat# PA1-10004; Anti-Neurofilament H (NF-H), Phosphorylated antibody: BioLegend Cat# 801602; Anti-beta-actin antibody: Novus Cat# NB100-56874; Anti-beta 3 Tubulin antibody: Santa Cruz Biotechnology Cat# sc-80005. Timed pregnant Sprague–Dawley rats were obtained from Central Animal Care Services at the University of Manitoba. Animals were housed under temperature-controlled conditions with a 12 hours light/dark cycle and ad libitum access to water and food. All efforts were made to minimize animal suffering and reduce the number of animals used.

Wang W., Liu Z., Jing B., Mai H., Jiao H., Guan T., Chen D., Kong J, & Pan T. (2022). 4,8-dicarboxyl-8,9-iridoid-1-glycoside Promotes Neural Stem Cell Differentiation Through MeCP2. Dose-Response, 20(3), 15593258221112959.

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Conjugation of EGF to Nanoparticles

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EGF Recombinant Human Protein (ThermoFisher, 1mg/mL, 1X PBS) was functionalized by adding 2% v/v of Propargyl-N-hydroxysuccinimidyl ester (Sigma-Aldrich, 0.44 M in DMSO, wt 225.20 g) and incubated on ice for 6 h. The functionalized EGF was then dialyzed in 1x PBS via D-Tube Dialyzers (Millipore) overnight to remove excessive NHS ester. The conjugation was completed by mixing 5 μL of functionalized EGF with membrane-wrapped NP (NP: EGF, 1:50000). The catalyst was prepared by mixing a 0.10 M solution of L-Ascorbic acid with a 5.10 mM solution of CuSO₄·5H₂O (1:1 v/v). And 3 μL of catalyst was added to the mixture of alkyne-functionalized EGF and azide-presenting NP. The final volume of the mixture was brought to 100 μL with 0.1x PBS. To generate NP-EGF with different EGF loading, membrane-wrapped NP with different wrapping times were used (Figure S5). The crosslinking reaction was maintained at room temperature overnight. Subsequently, excessive EGF and catalyst were removed by dialysis with Nuclepore Track-Etched Membranes (Whatman, Pore Size 0.03 μm, Diameter 25 mm) in 0.1x PBS overnight.

Zhang Q, & Reinhard B.M. (2018). Ligand Density and Nanoparticle Clustering Cooperate in the Multivalent Amplification of Epidermal Growth Factor Receptor Activation. ACS nano, 12(10), 10473-10485.

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Breast Cancer Spheroid Culture and Enrichment

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MCF-7 and SK-BR-3 breast cancer cells were purchased from the American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) according to standard protocols. Mammospheres are one of the most commonly used breast cancer spherical models and are able to enrich CD44⁺/CD24^−/low BCSLCs. MCF-7 (1×10³ cells/ml) and SK-BR-3 cells (1×10⁴ cells/ml) were cultured in suspension in serum-free DMEM-F12 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with B27 (1:50; Gibco; Thermo Fisher Scientific, Inc.; cat. no. 17504-044), 20 ng/ml EGF recombinant human protein (Gibco; Thermo Fisher Scientific, Inc.; cat. no. PHG0311), 0.4% BSA (Sigma-Aldrich; Merck KGaA), 4 mg/ml insulin (Sigma-Aldrich; Merck KGaA) and penicillin-streptomycin (100X; Invitrogen; Thermo Fisher Scientific, Inc.). The cells were shaken twice a day, 20 times each time, to prevent adhesion to the flask walls. Mammospheres was apparent after 7–10 days (30 (link),31 (link)).

Liu M.Y., Su H., Huang H.L, & Chen J.Q. (2019). Cancer stem-like cells with increased expression of NY-ESO-1 initiate breast cancer metastasis. Oncology Letters, 18(4), 3664-3672.

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Hydrogel-based Cell Culture Platform

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Dibenzocyclooctyne-sulfo-N-hydroxysuccinimidyl ester (DBCO-sulfo-NHS), dimethyl sulfoxide (DMSO), sodium hydroxide solution (1.0 N), agar, insulin, Triton-X, trypan blue solution, hyaluronidase type VI-S, collagenase type I, and resazurin based in vitro toxicology assay kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) pH 7.4, 10× PBS, Slide-A-Lyzer dialysis kit (3.5k MWCO), collagen I bovine protein solution (5 mg mL⁻¹), epidermal growth factor (EGF) recombinant human protein, fetal bovine serum (FBS), keratinocyte-serum free media (KSFM), bovine pyruvate extract (BPE), ITS Premix Universal Culture Supplement, trypsin, live/dead viability/cytotoxicity staining kit, paraformaldehyde (PFA), 5% normal goat serum, Alexa Fluor Phalloidin 488, Alexa Fluor 647-N-hydroxysuccinimidyl ester, and Alexa Fluor 546 secondary antibody were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Azido-poly(ethylene glycol)₅-N-hydroxysuccinimidyl ester (azido-PEG5-NHS) was purchased from BroadPharm (San Diego, CA, USA). Hyaluronate amine (40–50% degree of substitute) was purchased from Creative PEGWorks (Durham, NC, USA).

Chen F., Le P., Fernandes-Cunha G.M., Heilshorn S.C, & Myung D. (2020). Bio-orthogonally crosslinked hyaluronate-collagen hydrogel for suture-free corneal defect repair. Biomaterials, 255, 120176.

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Cytoskeleton Modulating Compounds in Cell Biology

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Thapsigargin was obtained from Cayman Chemical (Ann Arbor, MI, United States). Colcemid was purchased from Merck (Darmstadt, Germany). Tubastatin A was obtained from BioVision (Milpitas, CA, United States). Nocodazole was purchased from Abcam (Cambridge, United Kingdom). Fura-2/AM was obtained from Invitrogen. EGF Recombinant Human Protein was purchased from Thermo Fisher (Thermo Fisher Scientific, Waltham, MA, United States). The antibodies used in this study are shown in Supplementary Table 3.

Huang Y.T., Hsu Y.T., Chen Y.F, & Shen M.R. (2021). Super-Resolution Microscopy Reveals That Stromal Interaction Molecule 1 Trafficking Depends on Microtubule Dynamics. Frontiers in Physiology, 12, 762387.

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Piperlongumine-induced Oxidative Stress and Inflammation

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Piperlongumine (PPL) was purchased from AK Scientific California, USA. EGF recombinant human protein was purchased from Thermo Fisher Scientific, USA. MTT, AO, EB, DAPI, DCFDA, JC-1, LPS from Escherichia coli, protease inhibitor cocktail, Poly-L-lysine solution, FragEL™ DNA Fragmentation Detection Kit, and Fluorescent-TdT Enzyme Assay Kit were procured from Sigma-Aldrich, USA. Protein A/G Magnetic Beads were purchased from Invitrogen, USA. IMQ cream, 5% w/w was purchased from Glenmark Pharmaceuticals India. Nitrocellulose membrane, ECL reagent, and protein dual marker were procured from Bio-Rad, USA. MILLIPLEX MAP Kit was obtained from Millipore, Germany. Enzyme-linked immunosorbent assay kits for IL-1β, IL-6, IL-17A, IL-22, TNF-α, and TGF-β were procured from Thermo Fisher Scientific, USA. All the chemicals used in the present study were pure and of analytical grade.

Thatikonda S., Pooladanda V., Sigalapalli D.K, & Godugu C. (2020). Piperlongumine regulates epigenetic modulation and alleviates psoriasis-like skin inflammation via inhibition of hyperproliferation and inflammation. Cell Death & Disease, 11(1), 21.

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