Brachyury

Manufactured by Abcam

Sourced in United Kingdom

Brachyury is a transcription factor that plays a key role in the formation of the mesoderm during embryonic development. It is an important marker for cells undergoing the epithelial-to-mesenchymal transition process.

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Lab products found in correlation

Triton x 100, by Merck Group (3 mentions) Odyssey infrared imaging system, by LI COR (2 mentions) Fluorescent microscope, by Leica (1 mentions) Lsm 510 meta confocal microscope, by Zeiss (1 mentions) Muc 1 clone df3, by Abcam (1 mentions) β catenin, by Abcam (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Paraformaldehyde (pfa), by Fujifilm (1 mentions) Hoechst 33342, by Merck Group (1 mentions) Vector alkaline phosphatase substrate kit 3, by Vector Laboratories (1 mentions) Alexa fluor 594 conjugated goat anti mouse immunoglobulin g, by Thermo Fisher Scientific (1 mentions) Foxa2, by Cell Signaling Technology (1 mentions) Cell lysis buffer, by Cell Signaling Technology (1 mentions) Odyssey 3, by LI COR (1 mentions) Spectramax microplate spectrophotometer, by Molecular Devices (1 mentions) β actin, by Abcam (1 mentions) Protein assay reagent, by Bio-Rad (1 mentions) Ab178566, by Abcam (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Anti-Brachyury (3 citations)

10 protocols using brachyury

Immunochemical Analysis of Carcinoma Cells

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Carcinoma cells were grown on coverslips and stained as previously described (7 (link)) with monoclonal antibodies recognizing CDK1 (Cell Signaling), brachyury (Abcam) and MUC1 (clone DF3, 1/50 dilution). Where indicated, tumor cells were incubated with LAK cells at 37° C for one hour, and stained for F-actin utilizing Alexa Fluor 488® phalloidin as per the manufacturers’ recommendation. Images were captured utilizing a Leica Fluorescent microscope. Confocal images were obtained utilizing a Zeiss LSM 510 META Confocal Microscope.
Lung tumor tissue arrays were purchased from US Biomax, Inc. Sections of paraffin-embedded, formalin-fixed tissues were tested with a brachyury (Abcam) or a CDK1 antibody (EMD Millipore) as previously described (9 (link)), and counterstained with hematoxylin.

Hamilton D.H., Huang B., Fernando R.I., Tsang K.Y, & Palena C. (2014). WEE1 inhibition alleviates resistance to immune attack of tumor cells undergoing epithelial-mesenchymal transition. Cancer research, 74(9), 2510-2519.

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Chordoma Protein Expression Assay

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Cells were treated with AR-A014418 (2.5, 5, 10, 20, 30 μM) or DMSO, as a control, for 24 h. Then protein expression in the chordoma cell lysates was assessed by standard sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. GAPDH expression was used as an internal control. GSK3β (1:500), phosphorylated GSK3β(Ser9) (1:500) and GAPDH (1:2000) antibodies were purchased from Santa Cruz (California, USA). β-catenin (1:800) and brachyury (1:5000) antibodies were purchased from Abcam (Cambridge, UK).

Yan X., Li Z., Li H., Liu P., Zhao Z., Cheng S., Wang Z, & Zhang Q. (2019). Inhibition Of Glycogen Synthase Kinase 3 Beta Suppresses The Growth And Survival Of Skull Base Chordoma Cells By Downregulating Brachyury Expression. OncoTargets and therapy, 12, 9783-9791.

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Alkaline Phosphatase and Immunofluorescence Analysis

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The cells were fixed with 3.7% paraformaldehyde (Wako) in PBS for 10 minutes at room temperature. After washing with PBS three times, the level of alkaline phosphatase (AP) activity was detected by using the Vector Alkaline Phosphatase Substrate kit III (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instructions.
For the immunofluorescence analysis, cells were fixed with PBS that contained 3.7% paraformaldehyde for 10 minutes at room temperature. After washing with PBS, the cells were blocked with PBS that contained 5% BSA (Sigma–Aldrich) and 0.1% Triton X‐100 (Sigma–Aldrich) for 45 minutes at room temperature. Then, they were incubated overnight at 4°C with a primary antibody T (Brachyury, 1:1000, ab20680; Abcam, Tokyo, Japan). Alexa Fluor 594‐conjugated goat anti‐mouse immunoglobulin G (1:500; Invitrogen Life Technologies) was used as the secondary antibody. The nuclei were stained with 1 μg/mL Hoechst 33342 (Sigma–Aldrich).

Kim N., Minami N., Yamada M, & Imai H. (2016). Immobilized pH in culture reveals an optimal condition for somatic cell reprogramming and differentiation of pluripotent stem cells. Reproductive Medicine and Biology, 16(1), 58-66.

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Xenograft Tumor Characterization in Mice

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Cells were injected subcutaneously into NOD/SCID mice (∼5 × 10⁶ cells per site). Tumors were processed for hematoxylin-eosin staining at 8 weeks postinjection, and 4-μm-thick paraffin sections were treated with xylol and rehydrated in alcohol. The epitopes were retrieved in citrate buffer at 100°C for 30 min. IHC was performed with the Histostain-Plus IHC Kit (MRBiotech) with the following antibodies: FOXA2 (Cell Signaling Technologies), BRACHYURY (Abcam), and β-TUBULIN (Santa Cruz Technologies). All animal experimental procedures were conducted in accordance with the local Animal Welfare Act and Public Health Service Policy and approved by the animal experiment review board of the Institute of Life Sciences at East China Normal University (protocol #AR2013/05006).

Zhang X., Li B., Li W., Ma L., Zheng D., Li L., Yang W., Chu M., Chen W., Mailman R.B., Zhu J., Fan G., Archer T.K, & Wang Y. (2014). Transcriptional Repression by the BRG1-SWI/SNF Complex Affects the Pluripotency of Human Embryonic Stem Cells. Stem Cell Reports, 3(3), 460-474.

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PD-L1 Expression in Chordoma Cell Lines

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Expression of PD-L1 protein was evaluated by Western Blot analysis. Protein lysates from chordoma cell lines were extracted using 1× Cell Lysis Buffer (Cell Signaling Technology, MA). The protein concentrations were determined using Protein Assay Reagents (Bio-Rad, CA) and a SPECTRAmax Microplate Spectrophotometer from Molecular Devices (Sunnyvale, CA). The primary antibodies for PD-L1 (1:1000 dilution), brachyury (1:1000 dilution), and β-actin (1:2,000 dilution) were purchased from Abcam, Santa Cruz, and Sigma-Aldrich, respectively. The secondary antibodies were bound to IRDye1 800CW or IRDye1 680LT (LI-COR Biosciences, NE). Western blots were carried out as previously described [52 (link)]. Normalization was performed using actin as an endogenous control. Membrane signals were scanned using an Odyssey infrared imaging system and analyzed using Odyssey 3.0 software (LI-COR Biosciences, NE). The protein levels were quantified with NIH Image J software.

Feng Y., Shen J., Gao Y., Liao Y., Cote G., Choy E., Chebib I., Mankin H., Hornicek F, & Duan Z. (2015). Expression of programmed cell death ligand 1 (PD-L1) and prevalence of tumor-infiltrating lymphocytes (TILs) in chordoma. Oncotarget, 6(13), 11139-11149.

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Immunofluorescence Staining of Stem Cells

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Control and met-IPSC were cultured on 8-well culture chambers were washed with PBS, fixed with 4% paraformaldehyde in PBS for 10-30 min, permeabilized with 0.2% Triton X-100 (Sigma, St. Louis, USA) in PBS and blocked in 10% serum. Primary antibodies were diluted in PBS 10% serum at the following concentrations: Brachyury (ab140661, 1:200, Abcam, Cambridge, UK), phospho-Met (3077S, 1:200, Ozyme, Saint-Cyr-l’École, France), PODXL (ab178566, 1:200, Abcam, Cambridge, UK), SIX2 (11562-1-AP, 1:200, Euromedex, Souffelweyersheim, France), and then washed in PBS. The samples were incubated with fluorescent secondary antibodies in antibody dilution buffer, then washed in PBS. Nuclei were labeled with DAPI (D9542, Sigma–Aldrich, St. Louis, USA) mounting medium. Visualization and capture were realized with a NIKON microscope (NIKON, Minato, Japan).

Hwang J.W., Desterke C., Féraud O., Richard S., Ferlicot S., Verkarre V., Patard J.J., Loisel-Duwattez J., Foudi A., Griscelli F., Bennaceur-Griscelli A, & Turhan A.G. (2019). iPSC-Derived Embryoid Bodies as Models of c-Met-Mutated Hereditary Papillary Renal Cell Carcinoma. International Journal of Molecular Sciences, 20(19), 4867.

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Immunofluorescence Characterization of Stem Cells

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The following primary antibodies were used for fluorescence microscopy experiments at 1:200 dilution and secondary antibodies at 1:400 dilution: WT1 (Abcam, ab89901), PAX8 (Proteintech, 21384-1-AP), TUBB4A (Abcam, ab1315), BRACHYURY (Abcam, ab20680), POU5F1 (Stemgent, 09-0023), Nanog (Stemgent, 09-0020), SOX2 (Stemgent, 09-0024), TRA-1-60 (Stemgent, 09-0010), TRA-1-81 (Stemgent, 09-0011), SSEA4 (Stemgent, 09-0006), CDX2 (Biocare Medical, CM226A), SIX2 (Proteintech, 11562-1-AP), FOXJ1 (Abcam, ab40869), CDH1 (R&D System, AF648), OVGP1 (SIGMA, HPA062205) and DAPI (Molecular Probes, D3571).

Yucer N., Holzapfel M., Jenkins Vogel T., Lenaeus L., Ornelas L., Laury A., Sareen D., Barrett R., Karlan B.Y, & Svendsen C.N. (2017). Directed Differentiation of Human Induced Pluripotent Stem Cells into Fallopian Tube Epithelium. Scientific Reports, 7, 10741.

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Immunocytochemistry Protocol for Stem Cell Markers

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Cells were fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich) for 15 min at room temperature. They were
permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 10 min, blocked with 5% goat serum (Sigma-Aldrich) in
PBS and incubated overnight with primary antibodies at 4ºC. The primary antibodies were detected after
incubation with secondary antibodies for 45 min at room temperature. The primary antibodies used in this study
were against OCT-4 (Abcam, Cambridge, MA, USA; 1:200), SSEA-4 (Abcam; 1:200), TRA-1-60 (Chemicon, Temecula,
CA, USA; 1:100), TRA-1-81 (Chemicon; 1:100), Nestin (Chemicon; 1:200), Brachyury (Abcam; 1:200),
alpha-fetoprotein (AFP, Chemicon; 1:200), CDX2 (Abcam; 1:200) and EOMES (Abcam; 1:200). The secondary
antibodies used in this study were FITC conjugated- goat anti-rabbit (Abcam; 1:200), FITC conjugated- rabbit
anti-goat (Abcam; 1:200), Alexa Fluor 568 goat anti-rabbit (Life Technologies; 1:500) and Cy3 conjugated- goat
anti-mouse (Chemicon; 1:200). Cells were counterstained for nuclei with the 4'-6-diamidino-2-phenylindole
(DAPI; Sigma-Aldrich; 1:1000).

RUNGSIWIWUT R., NUMCHAISRIKA P., AHNONKITPANIT V., VIRUTAMASEN P, & PRUKSANANONDA K. (2016). Triploid human embryonic stem cells derived from tripronuclear zygotes displayed pluripotency and trophoblast differentiation ability similar to the diploid human embryonic stem cells. The Journal of Reproduction and Development, 62(2), 167-176.

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Tumor Biopsy Protein Analysis

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Commercial lysates from human lung and breast tumor biopsies were purchased from Imgenex (San Diego, CA). The following antibodies were used: pan-actin (clone Ab-5, Neo Markers), Beta-2 microglobulin (clone BBM.1, Santa Cruz), caspase 8 (clone Ab-3, Calbiochem), brachyury, lamin B1, lamin A/C (Abcam), CDK1 and caspase-3 (Cell Signaling Technology). Where indicated, protein lysates were run on a 7% acrylamide gel supplemented with 25 μM Phos-Tag (Wako Pure Chemical Industries), following the manufacture’s recommendations. All western blots were imaged and quantified using the Odyssey Infrared imaging system (LI-COR Biotechnology).

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Proteomic Analysis of NSCLC Cell Lines

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Proteins were isolated from the four human NSCLC epithelial cell lines using cell lysis buffer as previously described^{44 (link)}. Quantification of proteins in cell lysates was conducted by Bradford Assay followed by SDS-PAGE gel electrophoresis. The following primary antibodies diluted were used for western blot analyses^{44 (link)}: (1) CXCL2 (1:1,000; Cat. No. ab91511, Abcam); (2) brachyury (1:30,000; Cat. No. 81694, Cell Signaling Technology); (3) E-cadherin (1:1,000; Cat. No. 3195, Cell Signaling Technology); (4) β-catenin (1:2,000; Cat. No. 9562, Cell Signaling Technology); and (5) actin (1:2,000; Cat. No. ab8229, Abcam). Proteins were visualized by chemiluminescence autoradiography.

Kim J., Xu Z, & Marignani P.A. (2021). Single-cell RNA sequencing for the identification of early-stage lung cancer biomarkers from circulating blood. NPJ Genomic Medicine, 6, 87.

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