Anti human e cadherin

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-human E-cadherin is a primary antibody product used in laboratory research. It specifically binds to the E-cadherin protein, which is a cell-cell adhesion molecule expressed in epithelial cells. This antibody can be used to detect and study the expression and localization of E-cadherin in various experimental systems.

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8 protocols using anti human e cadherin

Western Blot Analysis of EMT and Cytokine Markers

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Cell lysate was prepared in radioimmunoprecipitation assay buffer lysis buffer
and debris completely removed by refrigerated centrifugation. The protein
concentration was determined by the bicinchoninic acid Protein Assay Kit
(ThermoFisher, MA, USA). An equal amount of protein was resolved by SDS-PAGE and
then transferred onto polyvinylidene difluoride (PVDF) membrane on ice. After
brief blocking with 5% skim milk in Tris-buffered saline with 0.1% Tween-20
(TBST), the PVDF membrane was hybridized with indicated primary Abs
(anti-human-E-cadherin, 1:1000, #14472; anti-human-N-cadherin, 1:1000, #14215;
anti-human-vimentin, 1:1000, #3390; anti-mouse-IL-6, 1:1000, #12912;
anti-mouse-TNF-α, 1:1000, #11948; anti-GAPDH, 1:1000, #2118; Cell Signaling
Technology, MA, USA) at 4°C overnight. The unbound Abs were washed off with TBST
next day and incubated with specific secondary Abs (anti-rabbit IgG, HRP-linked
Ab, 1:5000, #7074; anti-mouse IgG, HRP-linked Ab, 1:5000, #7076; Cell Signaling
Technology, MA, USA) at room temperature (25°C) for 1 h. The PVDF membrane was
then rigorously washed with TBST for 30 min and target protein bands were
visualized using the Enhanced Chemiluminescence Kit (Millipore, UT, USA) in
accordance with the manufacturer’s instructions. The endogenous GAPDH was
employed as loading control.

Wan G., Xie M., Yu H, & Chen H. (2018). Intestinal dysbacteriosis activates tumor-associated macrophages to promote epithelial-mesenchymal transition of colorectal cancer. Innate Immunity, 24(8), 480-489.

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Western Blot Analysis of EMT Markers

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The KFs with different treatment were harvested by centrifugation and washed with PBS. Cells were lysed in RIPA buffer containing protease inhibitors. The lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to Polyvinylidene Fluoride (PVDF) membranes by electroblotting. The membranes were incubated overnight at 4 °C with the primary antibodies after blocking with 5% nonfat milk. The anti-human E-cadherin, anti-human vimentin, anti-human PKM2, anti-human HIF-1α, and anti-human phospho-p70s6k primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The blots were then incubated with the secondary antibody for 2 h at room temperature. Each experiment was repeated for 3 times. Specific proteins were visualized using the ECL system (GE Healthcare) and the FUJIFILM Luminescent Image Analyzer LAS3000 (Fuji Film).

Lei R., Zhang S., Wang Y., Dai S., Sun J, & Zhu C. (2019). Metformin Inhibits Epithelial-to-Mesenchymal Transition of Keloid Fibroblasts via the HIF-1α/PKM2 Signaling Pathway. International Journal of Medical Sciences, 16(7), 960-966.

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Immunofluorescence Analysis of EMT Markers

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Long‐term estrogen depletion‐resistant MCF‐7 cells were seeded on cover slips and allowed to adhere overnight. After incubation with DMSO or EVE (100 nm) for 72 h, the cells were fixed with 4% paraformaldehyde in PBS, permeabilized in 0.1% Tween 20/PBS, and blocked with 1% BSA. The cells were washed and incubated with anti‐human E‐cadherin (1 : 100 dilution), cytokeratin 19 (CK19, 1 : 100 dilution), and ZEB1 (1 : 50 dilution) antibodies (all obtained from Cell Signaling Technology, Inc.) for 2 h at room temperature. The cells were subsequently washed and incubated with Alexa488‐labeled anti‐rat secondary antibody (Invitrogen, Carlsbad, CA, USA) for 2 h. After staining the nuclei with DAPI, the cells were visualized using a fluorescence microscope, BZ‐9000 (Keyence Corporation, Osaka, Japan).

Nishio E., Hayashi T., Akaza M., Hisatomi Y., Hikichi M., Fujii T., Utsumi T., Harada N, & Shimono Y. (2020). Upregulation of CIP2A in estrogen depletion‐resistant breast cancer cells treated with low‐dose everolimus. FEBS Open Bio, 10(10), 2072-2080.

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Western Blot Analysis of EMT Markers

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The cells were harvested by centrifugation and washed with PBS. The cells were lysed in RIPA buffer containing protease inhibitors. Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to Polyvinylidene Fluoride (PVDF) membranes by electroblotting. The membranes were blocked with 5% nonfat milk in Tris-buffered saline/0.1% Tween 20 for 1 h at room temperature and then incubated overnight at 4 °C with the primary antibodies. The anti-human E-cadherin, anti-human vimentin, anti-human SNAILl2, anti-human STAT3, and anti-human phospho-p70s6k primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-human β-actin primary antibodies and Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit and HRP-conjugated goat anti-mouse secondary antibodies were purchased from ZSGB-BIO (Beijing, China). After incubation with the secondary antibody for 1 h at room temperature, the protein bands were detected using the ECL detection system (BD Biosciences, New York, NY, USA).

Cheng K, & Hao M. (2016). Metformin Inhibits TGF-β1-Induced Epithelial-to-Mesenchymal Transition via PKM2 Relative-mTOR/p70s6k Signaling Pathway in Cervical Carcinoma Cells. International Journal of Molecular Sciences, 17(12), 2000.

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Immunofluorescent Labeling of E-cadherin

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After fixing in 4% paraformaldehyde, the cells were permeabilized with 0.1% Triton X-100 in PBS 3 times (5 min each) and incubated in blocking solution for 30 min at room temperature. After blocking, the cells were washed with PBS 3 times (5 min each) and incubated in anti-human E-cadherin (Cell Signaling Technology, Danvers, USA) overnight at 4°C. Then, the cells were washed 3 times with PBS, incubated for 30 min with Alexa Fluor 488-labeled Goat Anti-Rabbit IgG (Abcam, Cambridge, UK), and washed another 3 times with PBS. Nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI) and the sections were visualized using a fluorescence microscope.

Tang L., Zhu L., Zhang W., Yang X., Chen Q., Meng Z., Liu J., Sun Y., Hu J., Ni Z, & Wang X. (2020). Qi-Xian Decoction Upregulated E-cadherin Expression in Human Lung Epithelial Cells and Ovalbumin-Challenged Mice by Inhibiting Reactive Oxygen Species-Mediated Extracellular-Signal-Regulated Kinase (ERK) Activation. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 26, e922003-1-e922003-13.

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Protein Expression Analysis in Cells

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Total protein from cells was lysed using M-PER Mammalian Protein Extraction Reagent (Thermo) supplemented with a protease inhibitor cocktail (Sigma, St Louis, MO, USA). Samples were denatured, and equal amounts of protein were subjected to SDS-PAGE, and then transferred to nitrocellulose membrane. After blocking with 5% non-fat milk in TBST for 60 min, membranes were incubated with primary antibody dissolved in 5% bovine serum albumin in TBST overnight at 4°C. The following primary antibodies were used: anti-human- E-cadherin (1:2000, 24E10; Cell Signaling Technology, Danvers, MA, USA), anti-human-Vimentin (1:2000, D21H3; Cell Signaling Technology), anti-human-Snail (1:1000, C15D3; Cell Signaling Technology), anti-human-Twist (1:1000; Cell Signaling Technology), anti-human-Zeb (1:1000, D80D3; Cell Signaling Technology), and anti-human-Slug (1:1000, C19G7; Cell Signaling). Human GAPDH (1:5000; KangChen, Shanghai, China) was used as an internal reference.

Han Y., Ye J., Wu D., Wu P., Chen Z., Chen J., Gao S, & Huang J. (2014). LEIGC long non-coding RNA acts as a tumor suppressor in gastric carcinoma by inhibiting the epithelial-to-mesenchymal transition. BMC Cancer, 14, 932.

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Western Blot Analysis of Protein Expression

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Total protein lysates extracted from samples were quantitated using BCA protein assay kit (Pierce, Rockford, IL). 20 μg total protein lysates extracted from samples were separated with 10% sodium dodecyl sulfate-polyacryl-amide gels and transferred to a polyvinylidene fluoridemembrane. The membrane was blocked with 3% BSA, followed by incubation with mouse monoclonal anti-human WIF-1 (Abcam, Cambridge, MA), rabbit polyclonal anti-human E-cadherin, β-catenin and vimentin (Cell Signaling Technology Inc., Danvers, MA), anti-human β-Actin antibody (Epitomics, Burlingame, CA). The membrane was then incubated with secondary horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibody (Jackson Immune Research Laboratories Inc., West Grove, PA), and visualized using Immobilon™ Western Chemiluminescent HRP substrate (Millipore). β-Actin was used as the loading control.

Ji D., Chen Z., Li M., Zhan T., Yao Y., Zhang Z., Xi J., Yan L, & Gu J. (2014). MicroRNA-181a promotes tumor growth and liver metastasis in colorectal cancer by targeting the tumor suppressor WIF-1. Molecular Cancer, 13, 86.

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Immunofluorescent Localization of E-Cadherin

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Cells were allowed to attach overnight to glass coverslips in 12-well plates. The calcium concentration of the media was adjusted to 1.2 mM and the cells were incubated for 48 h. Cells were fixed with 4% formaldehyde for 10 min and washed in PBS. To detect E-cadherin, cells were blocked with 5% goat serum and incubated with a rabbit monoclonal anti-human E-cadherin (24E10, Cell Signaling Technology, Beverley, MA) for 2 h at room temperature. Bound primary antibody was detected using an Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen Corporation) and the nuclei were counterstained with TOPO-3 (Invitrogen Corporation). Sections were examined by confocal microscopy.

YUAN F.N., VALIYAPARAMBIL J., WOODS M.C., TRAN H., PANT R., ADAMS J.S, & MALLYA S.M. (2014). Vitamin D signaling regulates oral keratinocyte proliferation in vitro and in vivo. International Journal of Oncology, 44(5), 1625-1633.

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