Nodal

Manufactured by R&D Systems

Sourced in Germany, United States

Nodal is a laboratory instrument designed for the analysis of cellular signaling pathways. It functions by detecting and quantifying the expression levels of specific proteins involved in these pathways. The core operation of Nodal involves the use of immunoassay techniques to measure the abundance of target proteins in cellular samples.

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Lab products found in correlation

Bmp 2, by R&D Systems (2 mentions) Bone morphogenetic protein 4 (bmp4), by R&D Systems (2 mentions) Stemmacs ips brew medium, by Miltenyi Biotec (1 mentions) Insulin, by Selleck Chemicals (1 mentions) Xav939, by Selleck Chemicals (1 mentions) Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (1 mentions) Chir99021, by Selleck Chemicals (1 mentions) Pd0325901, by Merck Group (1 mentions) Ml347, by Selleck Chemicals (1 mentions) Ldn193189, by Bio-Techne (1 mentions) Pd98059, by Merck Group (1 mentions) 8 br camp, by Bio-Techne (1 mentions) Sb431542, by Bio-Techne (1 mentions) Zstk474, by Selleck Chemicals (1 mentions) Noggin, by Thermo Fisher Scientific (1 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions) Retinoic acid, by Merck Group (1 mentions) Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions) Clariostar reader, by BMG Labtech (1 mentions) Grp78, by Abcam (1 mentions)

Close spelling variants of this product

Recombinant human nodal (2 citations) Recombinant NODAL (2 citations)

10 protocols using nodal

Modulation of hiPSC Differentiation

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Undifferentiated hiPSCs were treated for 2 h with recombinant NODAL (R&D, 3218-ND-025) at concentrations ranging from 10 to 200 ng/mL in StemMACS iPS-Brew medium without supplements (Miltenyi).
Undifferentiated hiPSCs were treated for 24 h with the wild-type PCSK9 recombinant protein (rPCSK9-WT; Circulex, CY-R2330) or the GOF D374Y-PCSK9 recombinant protein (rPCSK9 D374Y; Circulex, CY-R2311) at a concentration of 600 ng/mL in the routinely used hiPSC culture medium, for each recombinant protein.

Roudaut M., Idriss S., Caillaud A., Girardeau A., Rimbert A., Champon B., David A., Lévêque A., Arnaud L., Pichelin M., Prieur X., Prat A., Seidah N.G., Zibara K., Le May C., Cariou B, & Si-Tayeb K. (2021). PCSK9 regulates the NODAL signaling pathway and cellular proliferation in hiPSCs. Stem Cell Reports, 16(12), 2958-2972.

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Compound Serial Dilution Assays

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Serial dilutions of compounds were made in appropriate solvents (DMSO or H₂O) and corresponding carrier controls were included in the assays. Serial dilutions were made for single soluble factor titrations; FGF4, sodium (ortho)vanadate (Sigma-Aldrich, S6508), PD0325901, PD98059 (Sigma, P215), activin A, TGFβ1, A83-01 (Tocris, 2939), Nodal (R&D systems, 1315-ND-025), SB431542 (Tocris, 1614), retinoic acid (Sigma-Aldrich, R2625), DL-epinephrine HCl (Sigma-Aldrich, E4642), 8Br-cAMP, SC144 (Tocris, 4963/10), IL6 (Peprotech, 216-16), IL11, Lif, BMP4 (Peprotech, 315-27), BMP7, LDN 193189 (Tocris, 6053), ML347 (Selleckchem, S7148), Noggin (Peprotech, 250-38), CHIR99021, IWP2 (Selleckchem, S7085) and XAV-939 (Selleckchem, S1180). Compounds with end concentrations that were used for XEn/Epi EpiC modulation: PI3K inhibitor ZSTK474 (Selleckchem, S1072) at 1 μM, insulin at 50 ng/ml, XAV-939 at 20 μM.

Vrij E.J., Scholte op Reimer Y.S., Fuentes L.R., Guerreiro I.M., Holzmann V., Aldeguer J.F., Sestini G., Koo B.K., Kind J., van Blitterswijk C.A, & Rivron N.C. (2022). A pendulum of induction between the epiblast and extra-embryonic endoderm supports post-implantation progression. Development (Cambridge, England), 149(20), dev192310.

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CR1-Binding Protein Interaction Assay

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CR1 (R&D Systems, Cat# 145-CR/CF), Nodal (R&D Systems, Cat# 3218-ND-024/CF), GRP78 (Abcam, Cat# ab78432), and Alk4 (Creative BioMart, Cat# ACVR1B-645H,) were purchased from the indicated vendors. CR1/BP interaction assays were run in parallel to that described previously by Klauzinska et al. (19 (link)). In brief, four different target proteins (Nodal, GRP78, ACVR1B/Alk4, and BSA) were solid phased to a 96-well plate (Costar, Cat# 3591, Polystyrene) at 50 ng/50 µl/well in two separate columns containing octuplicate wells for each target protein, agitated for 2 h on a rotary mixer and transferred to 4 °C overnight. Then, half the target columns were exposed to 25 µl 1% BSA/PBS (Negative Control–100% CR1 binding), and the other half of the target columns were exposed to 25 µl SFN [100 µM]. All the plates were treated with 25 µl CR1 [50 ng/well], 50 µl rabbit anti-N-terminal CR1 (Abcam, Cat# ab103891), and 50 µl goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Cat# sc-2004) according to the procedure published previously (19 (link)) – a PBS wash and 30min room temperature rotary shaker incubation was performed between each treatment following CR1 addition. Finally, stop solution (50 µl) was added to all wells and the plates were assessed for the absorbance at 450 nm on a CLARIOstar reader (BMG Labtech). Data was evaluated using Microsoft Excel program.

Castro N.P., Rangel M.C., Merchant A.S., MacKinnon G., Cuttitta F., Salomon D.S, & Kim Y.S. (2019). Sulforaphane suppresses the growth of triple-negative breast cancer stem-like cells in vitro and in vivo. Cancer prevention research (Philadelphia, Pa.), 12(3), 147-158.

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Pharmacological Modulation of Morphogen Signaling

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The pharmacologic agents dabrafenib and trametinib (both from Hycultec, Beutelsbach, Germany), alone or in combination with the morphogens, were added directly to the culture medium of the cells in monolayer at concentrations previously shown to be effective in single agent experiments. Cells treated with culture medium or culture medium with the addition of DMSO served as controls. For all experiments, we used the agonists and antagonists in the following concentrations: BMP-2 (20 ng/ml), BMP-7 (30 ng/ml), noggin (100 ng/ml), nodal (30 ng/ml), lefty (100 ng/ml) (all from R&D Systems, Wiesbaden, Germany), and the ALK4/5/7 nodal/activin receptor antagonist SB431542 (30 µM, Sigma-Aldrich).

Sinnberg T., Niessner H., Levesque M.P., Dettweiler C., Garbe C, & Busch C. (2018). Embryonic bone morphogenetic protein and nodal induce invasion in melanocytes and melanoma cells. Biology Open, 7(6), bio032656.

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Directed Differentiation of Stem Cells

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When cells had reached confluency (typically 15–20 h after seeding), germ layer differentiation was induced using N2B27 medium consisting of 93% DMEM, 1% penicillin-streptomycin, 0.1 mM β-mercaptoethanol, 1% non-essential amino acids, 1% Glutamax, 2% B27 minus retinoic acid, 1% N2 supplement, and supplemented with 50 ng/mL BMP4 (R&D), 100 ng/mL NODAL (R&D), and 10 ng/mL bFGF (Thermo Fisher). Cells were incubated for 48 h at 37.5°C prior to fixation.

Vickers A., Tewary M., Laddach A., Poletti M., Salameti V., Fraternali F., Danovi D, & Watt F.M. (2021). Plating human iPSC lines on micropatterned substrates reveals role for ITGB1 nsSNV in endoderm formation. Stem Cell Reports, 16(11), 2628-2641.

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Western Blot Analysis of Signaling Pathways

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Both INS-1 cells and rat islets were lysed in RIPA lysis buffer, 25 μg of protein was loaded and resolved by SDS-PAGE followed by semidry transfer to nitrocellulose membranes [9 ]. The primary antibodies used were as follows: Smad3 (1:1000), phosphorylated Smad3 (p-Smad3) (1:1000), Akt (1:1,000), p-Akt (1:1,000), ERK1/2 (1:1,000), p-ERK1/2 (1:1,000), β-catenin (1:1,000), p-β-catenin (Ser-675) (1:1,000), caspase-3 (1:1,000) and cleaved caspase-3 (1:1,000) were obtained from Cell Signaling (Danvers, MA, USA); Nodal (1:500) and ALK7 (1:1,000) were from R&D Systems; and GSK-3β (1:1,000), p-GSK-3β (1:1,000) as well as GAPDH (1:10,000) were from Abcam (Cambridge, MA, USA). Protein band densities were quantified using the ImageJ program, and data were normalized to control [9 ].

Li J., Wang Z., Ren L., Fan L., Liu W., Jiang Y., Lau H.K., Liu R, & Wang Q. (2018). Antagonistic interaction between Nodal and insulin modulates pancreatic β-cell proliferation and survival. Cell Communication and Signaling : CCS, 16, 79.

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Esophageal Cell Lines and Treatments

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The Barrett's esophagus cell line CPB (CRL-4028) was purchased from American Type Culture Collection (ATCC) and cultured with epithelial cell medium 2 (ScienCell, Carlsbad, CA) supplemented with 5% fetal bovine serum (FBS, Hyclone, GE Healthcare, Pittsburgh, PA) and antibiotics, 100 units/mL penicillin and 100 μg/mL streptomycin (Gibco, Carlsbad, CA). The esophageal adenocarcinoma cell lines, OE33 and FLO-1, were derived by Dr. David Beer [60 (link)] and grown in RPMI and DMEM (Invitrogen, Carlsbad, CA), respectively, with 10% FBS at 37°C in 5% CO₂. Fibroblasts were grown in DMEM with 5% FBS (Hyclone), 100 units/mL penicillin, and 100 μg/mL streptomycin (Gibco). For treatment with growth factors 5 ng/ml recombinant human TGFβ1, 10 ng/ml Activin A, 100 ng/ml Follistatin-288, 100 ng/ml Nodal (all R&D Systems, Minneapolis, MN Systems) or 1 μM A83-01 (Tocris, Bristol, UK) were used. Overexpression of Activin A (INHBA) was achieved by retroviral transfection of cells with viral supernatant containing pBABE plasmid with zeocin resistance (Addgene, Cambridge, MA) encoding the INHBA gene sequence (Origene, Rockville, MD).

Taylor C., Loomans H.A., Le Bras G.F., Koumangoye R.B., Romero-Morales A.I., Quast L.L., Zaika A.I., El-Rifai W., Andl T, & Andl C.D. (2015). Activin a signaling regulates cell invasion and proliferation in esophageal adenocarcinoma. Oncotarget, 6(33), 34228-34244.

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Compound-based Cell Treatment Protocol

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Compounds and growth factors used to treat cells were: Rapamycin, TGFβ, HGF (EMD Millipore, Billerica, MA), Activin (eBioscience, San Diego, CA), BMP4, Nodal, Torin1 (R&D Systems, Minneapolis, MN), Insulin, Transferrin, and Selenium (ITS) (Roche, San Francisco, CA), SU11274, AG490 (Sigma Aldrich, St. Louis, MO), and CGP57380 (Cayman Chemicals, Ann Arbor, MI). U0126 was obtained from Promega (Madison, WI). A TGFβ Type I Receptor kinase inhibitor (616451) was purchased from EMD Millipore (Billerica, MA).

Jahn S.C., Law M.E., Corsino P.E., Davis B.J., Harrison J.K, & Law B.K. (2014). Signaling Mechanisms that Suppress the Cytostatic Actions of Rapamycin. PLoS ONE, 9(6), e99927.

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Astrocyte Survival Assay with Cytokines

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Acutely isolated astrocytes were plated at 2,000 cells/well in poly-d-lysine-coated (PDL) 24-well plates in base media with 0.5 µg/ml aphidicolin (Sigma A0781). Unless otherwise noted, candidate cytokines were individually added at the following concentrations: 5 ng/ml HbEGF (Peprotech); 100 ng/ml IGF2 (R&D Systems); 100 ng/ml CXCL12 (R&D Systems); 50 ng/ml BDNF (Peprotech); 10 ng/ml NT3 (Peprotech); 10 ng/ml FGF1, FGF2, FGF8, and FGF9 (Peprotech); 100 ng/ml BMP2, BMP4, BMP5, BMP6, BMP7, BMP10, BMP15, GDF3, and GDF5 (R&D Systems); 250 ng/ml Activin A (R&D Systems); 100 ng/ml Nodal (R&D Systems); 100 ng/ml TGFB2 (R&D Systems). Survival was assayed at 3DIV using a Live/Dead kit in which calcein AM stains live cells green and ethidium homodimer stains dead cells red (Life Technologies L3224). At least 3 independent experiments were conducted for each condition. For each experiment, 3 non-overlapping 20x fields per well were quantified in triplicate wells. Significance determined using one-way ANOVA with Dunnett correction.

Scholze A.R., Foo L.C., Mulinyawe S, & Barres B.A. (2014). BMP Signaling in Astrocytes Downregulates EGFR to Modulate Survival and Maturation. PLoS ONE, 9(10), e110668.

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Isolation and Culture of Tumor-Initiating Cells

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Tumor tissue was minced into small pieces (< 2 mm) and digested with 2 mg/ml collagenase IV in Medium 199 (Invitrogen) containing 3 mM CaCl₂ for 2.5 h. Subsequently, purified tumor cells were washed with D‐PBS (Invitrogen) and filtered once though a 100‐μm and twice through a 40‐μm cell strainer (BD Biosciences) to receive a single‐cell suspension. For serum‐free culture conditions, DMEM/F12 medium was used supplied with 6 mg/ml glucose, 2 mM l‐glutamine, 1% penicillin/streptomycin, B27 supplement (Invitrogen), 5 mM HEPES buffer, 6 μg/ml heparin (Sigma‐Aldrich), 10 ng/ml FGF2, 20 ng/ml FGF10, and 20 ng/ml Nodal (R&D Systems). To establish spheroid cultures, purified single‐cell suspensions were taken into serum‐free medium in ultra‐low attachment plates (Corning). For adherent cultures, undigested whole‐tumor pieces were plated into normal cell culture flasks (Thermo scientific) to allow attachment of tumor cells. For phenotypic differentiation, adherent cells were detached by Accutase (PAA) and replated in RPMI 1640 medium supplemented with 2 mM l‐glutamine, 1% penicillin/streptomycin (Invitrogen), and 10% fetal bovine serum (FBS) (PAA). Cultures were split 1:1 to 1:5 by Accutase (PAA)‐mediated cell detachment. Cytokines were added every 3–4 days to serum‐free cultures. Established primary TIC cultures were regularly tested mycoplasma‐free.

Ball C.R., Oppel F., Ehrenberg K.R., Dubash T.D., Dieter S.M., Hoffmann C.M., Abel U., Herbst F., Koch M., Werner J., Bergmann F., Ishaque N., Schmidt M., von Kalle C., Scholl C., Fröhling S., Brors B., Weichert W., Weitz J, & Glimm H. (2017). Succession of transiently active tumor‐initiating cell clones in human pancreatic cancer xenografts. EMBO Molecular Medicine, 9(7), 918-932.

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