14-9326-82

antibody from Invitrogen Antibodies

Targeting: EPCAM 17-1A, 323/A3, CD326, CO-17A, EGP-2, EGP34, EGP40, Ep-CAM, ESA, GA733-2, HEA125, KS1/4, KSA, Ly74, M4S1, MH99, MIC18, MK-1, MOC31, TACST-1, TACSTD1, TROP1

Provider product page for 14-9326-82

Western blot

Immunoprecipitation

Antibody data

Antibody Data
Antigen structure
References [23]
Comments [0]
Validations
Immunocytochemistry [2]
Immunohistochemistry [2]
Flow cytometry [1]
Other assay [21]

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Product number: 14-9326-82 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD326 (EpCAM) Monoclonal Antibody (1B7), eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: EpCAM (Epithelial cell adhesion molecule, CD326, KSA, TROP1) is a 40 kD cell-surface adhesion molecule participating in homophilic, calcium-independent cell-cell interactions. EpCAM is a type-I transmembrane protein, and is expressed primarily on the basolateral surface of most epithelia. Although normal epithelia express low levels of EpCAM, increased expression has been correlated with increased proliferation and progression to a mesenchymal phenotype. EpCAM has also been used as a diagnostic marker on circulating metastatic carcinoma cells, while cancer cells of non-epithelial origin do not express EpCAM. Applications Reported: This 1B7 antibody has been reported for use in flow cytometric analysis, immunohistochemistry of formalin-fixed paraffin embedded or frozen tissue sections, and immunoprecipitation. The 1B7 antibody has also been found useful for immunoblotting, and recognizes a protein of approximately 40 kD under non-reducing conditions. Applications Tested: This 1B7 antibody has been tested by flow cytometric analysis of A549 human lung carcinoma cells. This can be used at less than or equal to 0.25 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. The 1B7 antibody has been tested by immunohistochemistry of formalin-fixed paraffin embedded human tissue using low or high pH antigen retrieval and can be used at less than or equal to 10 µg/mL. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. Purity: Greater than 90%, as determined by SDS-PAGE. Aggregation: Less than 10%, as determined by HPLC. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: 1B7
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: 4° C

EPCAM protein structure - 14-9326-82 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of EpCAM was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR701274_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Immunofluorescence analysis was performed on wild type A-431 cells (panel a,d), A-431 Cas9 control cells (panel b,e) and A-431 EpCAMKO cells (panel c, f). Cells were fixed, permeabilized, and labeled with EpCAM Monoclonal Antibody (Ber-EP4) (Product # 14-9326-82, 5 µg/mL dilution), followed by Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (Product # A32723, 1:2000 dilution). Nuclei (blue) were stained using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Loss of signal (panel c,f) upon CRISPR mediated knockout (KO) confirms that antibody is specific to EpCAM (green). The images were captured at 60X magnification.

Supportive validation

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Experimental details: Immunofluorescence analysis of EpCAM was performed using HT-29 and BJ cells. The cells were fixed with 4% paraformaldehyde for 10 minutes and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with EpCAM Mouse Monoclonal Antibody (Product # 14-9326-82) at 5 µg/mL in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green) in HT-29 cells. Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image of HT-29 cells, which is a positive model for EpCAM expression showing a plasma membrane localization. Panel e represents the merged image of BJ cells, that are null for EpCAM protein expression. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

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Supportive validation

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Supportive validation

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Experimental details: 2 Figure H. pylori activate PI3K/AKT/mTOR/S6K pathway in gastric epithelial cells in a cagA -dependent manner. (a) Representative immunofluorescence staining images showing p-mTOR-expressing (red) CD326 + gastric epithelial cells (green) in gastric mucosa of H. pylori -infected patients. Scale bar = 100 um. (b) Representative immunofluorescence staining images showing p-mTOR expression in WT H. pylori -infected, DeltacagA -infected, and uninfected AGS cells. Red, p-mTOR; blue, DAPI-stained nuclei. Scale bar = 10 um. (c and d) Representative data (c) and statistical analysis (d) of p-mTOR and mTOR proteins in H. pylori -infected and uninfected AGS cells at a different time point (MOI = 100) or with different MOI (3 hr) analyzed by western blot were shown ( n = 3). (e and f) Representative data (e) and statistical analysis (f) of p-AKT and AKT proteins, p-mTOR and mTOR proteins, p-S6K and S6K proteins in WT H. pylori -infected, DeltacagA -infected, and uninfected AGS cells (MOI = 100, 3 hr) or in WT H. pylori -infected AGS cells (MOI = 100, 3 hr) pretreated with Wortmannin or rapamycin analyzed by western blot were shown ( n = 3). (e and f) Representative data (e) and statistical analysis (f) of p-AKT and AKT proteins, p-mTOR and mTOR proteins, p-S6K and S6K proteins in WT H. pylori -infected, DeltacagA -infected, and uninfected human primary gastric epithelial cells (MOI = 100, 3 hr) or in WT H. pylori -infected human primary gastric epithelial cells (MOI = 100, 3 hr) pretreat

Supportive validation

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Experimental details: Fig. 5 FZD5 maintains stem cell-like properties. A CD133, EPCAM, ALDH1A2, and POU5F1 expression in MDA-MB-231 cells was detected by real-time PCR. B Fractions of CD133-positive and EPCAM-positive MDA-MB-231 cells were detected by flow cytometry. C Mammosphere formation of MDA-MB-231 cells was shown. Scale bar: 500 mum, mean +- SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, vs. shCtrl. D CD133, EPCAM, ALDH1A2, and POU5F1 expression in Hs-578t cells was detected by real-time PCR. E Fractions of CD133-positive and E PCAM-positive Hs-578t cells were detected by flow cytometry. F Mammosphere formation of Hs-578t cells was shown. Scale bar: 500 mum, mean +- SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, vs. OE Ctrl.

Supportive validation

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Experimental details: Figure 1 Multiparametric flow cytometry of PBMCs capturing uPAR/int beta1 CTC subsets. Breast cancer PBMCs were first sorted applying gating parameters to select for DAPI - (4', 6-diamidino-2-phenylindole)/EpCAM - /CD45 - /CD44 + /CD24 - cells. Cells were then subsequently sorted to obtain uPAR/int beta1 subsets containing combinatorial expression of these markers. Antibodies used for flow cytometry and cell sorting were: anti-human CD45-APC-Cy7 (Biolegend, cat # 304015, 1:50 dilution), mouse anti-human EpCAM-PE CD326 (eBiosciences, cat # 12-9326-71, 1:40 dilution), anti-human CD24-PE ML5 (Biolegend, cat # 311106, 1:20 dilution), anti-human CD44-PE-Cy7 IM7 (Biolegend, cat # 103030, 1:20 dilution), mouse anti-human uPAR (CD87)-FITC (AbD Serotec cat # MCA2506FT, 1:10 dilution), anti-human int beta1 (CD29)-ApC TS2/16 (Biolegend, cat # 3030008, 1:50 dilution). Cells were confirmed to be CTCs by performing RT-PCR, immunoflurescence and genotyping arrays. Representative images are shown.

Supportive validation

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Experimental details: Figure 1 LGR 5- EGFP and KI 67-Tag RFP 2 knock-in PDO s Design of LGR5-EGFP donor and CRISPR/Cas9 sgRNA vectors. Blue circle represents the CRISPR/Cas9 protein complex and the yellow box underneath the guide RNA. Flow cytometry profiles at day 20 post-nucleofection. Immunofluorescence for DAPI, EGFP, and KRT20 or MUC2 in in vitro cultured PDO#7-LGR5-EGFP#1. Scale bars indicate 100 mum. FACS profiles showing EGFP-high (green), -low (blue), and -negative (gray) cells in PDO#7-LGR5-EGFP#1 and #2 organoids. Relative mRNA expression level by real-time qPCR in cells expressing distinct levels of EGFP isolated from PDO#7-LGR5-EGFP#1 and #2 knock-in organoids. Values show mean +- s.d. of three measurements. Design of KI67-TagRFP2 donor and CRISPR/Cas9 sgRNA vectors. Blue circle represents the CRISPR/Cas9 protein complex and the yellow box underneath the guide RNA. Images of PDO#7-KI67-TagRFP2#1 organoids. Scale bars indicate 100 mum. PDO#7-KI67-TagRFP2#1 xenograft. TagRFP2 co-localizes with DAPI nuclear staining. Scale bars indicate 25 mum. Flow cytometry analysis of EPCAM + /DAPI - cell population of PDO#7-LGR5-EGFP/KI67-TagRFP2#1 from disaggregated xenografts. Cell cycle analysis of KI67-TagRFP2-positive and KI67-TagRFP2-negative cells from PDO#7-LGR5-EGFP/KI67-TagRFP2#1 disaggregated xenografts. X -axis shows DNA content and y -axis EdU incorporation. Quantification of the frequencies of KI67 + versus KI67 - cells found in each cell cycle phase.

Supportive validation

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Experimental details: Figure 8 Elevated IL-17RB expression in cancer cells derived from tumor-draining lymph nodes in breast cancer patients correlates with aggressive growth nature A Freshly collected human breast cancer (hBC) specimens (PT, primary tumors; LN, paired LN metastasis from the same breast cancer patient) were digested with collagenase-containing buffer overnight. Depletion of hematopoietic cells by CD45 Dynabeads (Invitrogen) enriched the EpCAM + cells for following xenografts. Percentages of EpCAM + cells in human primary breast cancer cells were confirmed by FACS analysis (left two panels: before enrichment; right two panels: after enrichment). All of the histograms were shown that they were EpCAM expressing (black line), compared to isotype control (gray filled). B Tumorigenesis assay was determined in NOD/SCID/IL2Rgamma mice orthotopically injected with hBC PT and hBC LN cells. Representative data on day 180 were shown. White arrow: Orthotopic injection site. C Dot plot illustrated the tumor volumes of hBC PT and hBC LN cell-injected NOD/SCID/IL2Rgamma mice on day 180 ( n = 11). All tumors were confirmed by pan-keratin IHC staining of fat pad sections. D, E H&E stainings and IHC stainings of pan-keratin were shown in the representative cases of fat pad sections from (B). Scale bar: 1 mm (left panels), 100 mum (right panels). F IHC stainings of IL-17RB were shown in the representative cases of fat pad sections from (B). Scale bar: 10 mum. G The correlatio

Supportive validation

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Experimental details: Fig. 4 EMP3 interferes with stem-like properties. A CCLE database was interrogated for EMP3, SOX2, CD133, and EPCAM expression. Correlation between two genes in a total of 57 breast cancer cell lines was analyzed by Pearson statistics. B SOX2, CD133, and EPCAM expression in MDA-MB-231 cells were detected by real-time PCR. Mean +- SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, vs. shCtrl. C Fractions of CD133-positive MDA-MB-231 cells were detected by flow cytometry. Mean +- SD, n = 3. ** P < 0.01, vs. shCtrl. D Fractions of EPCAM-positive MDA-MB-231 cells were detected by flow cytometry. Mean +- SD, n = 3. ** P < 0.01, vs. shCtrl. E Mammosphere formation of MDA-MB-231 cells was shown. Mean +- SD, n = 3. ** P < 0.01, *** P < 0.001, vs. shCtrl. F Mammosphere formation of MCF7 cells was shown. Mean +- SD, n = 3. ** P < 0.01, vs. OE Ctrl. G SOX2, CD133, and EPCAM expression in MCF7 cells were detected by real-time PCR. Mean +- SD, n = 3. *** P < 0.001, vs. OE Ctrl.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Generation of proximal lung organoids from cystic fibrosis patient iPSCs (A) Schematic representation of lung organoid differentiation protocol from human iPSCs (hiPSCs). (B) Representative immunofluorescence staining for p63 (green), NKX2.1 (red), SOX2 (red), and nuclear protein expression with DNA stain (DAPI; blue) in HLOs on day 20 (p63 and NKX2.1) and day 22 (SOX2). Scale bars represent 100 mum. (C) Flow cytometry analysis of EpCAM expression in organoid cells on day 23. Left: summary percentage of EpCAM-positive cells. Three independent HLO plates were used for the experiment. Data are presented as mean +- standard error of the mean (SEM). Right: a representative flow cytometry result. The x axis indicates the count of cells, and the y axis indicates EpCAM signal level.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 1 Identification of three major epithelial cell types and their markers using scRNAseq. a Overview of scRNAseq approach using primary human breast tissue samples that were processed into single cell suspension, followed by FACS isolation of basal (CD49f-hi, EPCAM+) and luminal (CD49f+, EPCAM-hi), and scRNAseq analysis using the microfluidics-enabled scRNAseq. b Combined tSNE projection of cells from all three microfluidics-enabled scRNAseq datasets. The major basal cluster is highlighted in red; Luminal1 (L1) in green; Luminal2 (L2) in blue. c Heatmap displaying the scaled expression patterns of top marker genes within each cell type with selected marker genes highlighted; yellow indicating high expression of a particular gene, and purple indicating low expression. d Feature plots showing the scaled expression of TCF4 and ZEB1 marking a subpopulation of basal cells and gene plot showing co-expression of TCF4 and ZEB1 in the same cells. See Supplementary Fig. 1 capture site imaging, gene detection, individual principal component analysis, tSNE plot colored by individual-derived cells and feature plots of cell type-specific markers

Supportive validation

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Experimental details: Figure 1 Characterization of BCSC5 in vitro and in vivo. ( A ) Representative pictures of BCSC5 cultured in 3D and 2D conditions, scale bar 100 mum. ( B ) Sphere-forming capacity of BCSC5 cells in an anchorage-independent growth assay ( n = 3). Data represent means + SEM. ( C , D ) Expression patterns of CD24 and CD44 ( C ) as well as EpCAM and CD49f ( D ) in BCSC5 cells analyzed by flow cytometry. ( E ) Tumor formation in limiting dilution xenografts of BCSC5. ( F ) Representative growth curves for limiting dilution assay of BCSC5 xenografts in immunocompromised NOD/SCID mice. ( G ) Immunohistochemical (IHC) analysis of ER, PR, and HER2 on sections of the BCSC5 xenograft tumors, scale bar 100 mum.