MA5-29246

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

Western blot (Supportive data in Antibodypedia)

ELISA (Recommended by provider)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunoprecipitation (Data presented on provider website)

Immunohistochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

MA5-29246

Provider

Invitrogen Antibodies

Product name

EpCAM Recombinant Rabbit Monoclonal Antibody (28)

Antibody type

Monoclonal

Antigen

Recombinant full-length protein

Description

This product is preservative free. It is recommended to add sodium azide to avoid contamination (final concentration 0.05%-0.1%). Recombinant rabbit monoclonal antibodies are produced using in vitro expression systems. The expression systems are developed by cloning in the specific antibody DNA sequences from immunoreactive rabbits. Then, individual clones are screened to select the best candidates for production. The advantages of using recombinant rabbit monoclonal antibodies include: better specificity and sensitivity, lot-to-lot consistency, animal origin-free formulations, and broader immunoreactivity to diverse targets due to larger rabbit immune repertoire. This antibody has specificity for Human EpCAM/TROP1/CD326.

Reactivity

Human

Host

Rabbit

Isotype

IgG

Antibody clone number

28

Vial size

100 µL

Concentration

1.22 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

References

Submitted references

High throughput, label-free isolation of circulating tumor cell clusters in meshed microwells.

Boya M, Ozkaya-Ahmadov T, Swain BE, Chu CH, Asmare N, Civelekoglu O, Liu R, Lee D, Tobia S, Biliya S, McDonald LD, Nazha B, Kucuk O, Sanda MG, Benigno BB, Moreno CS, Bilen MA, McDonald JF, Sarioglu AF
Nature communications 2022 Jun 13;13(1):3385

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of EpCAM in Lane A: MCF-7 Whole Cell Lysate (30 µg). Samples were probed using a EpCAM Monoclonal Antibody (Product # MA5-29246) at a 1:500 dilution, followed by a Goat Anti-Rabbit IgG (H+L), Dylight 800 Secondary Antibody at a 1:10000 dilution. Western blot was performed under reducing conditions. Predicted band size:34 kDa. Observed band size:38 kDa.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western Blot using EpCAM Recombinant Rabbit Monoclonal Antibody (28) (Product # MA5-29246) at 1:500 dilution. Lane A: MCF-7 Whole Cell Lysate. Lysates/proteins at 30 μg per lane. Secondary Goat Anti-Rabbit IgG H&L (DyLight™ 800) at 1:10,000 dilution. Developed using the Odyssey technique. Performed under reducing conditions. Predicted band size: 34 kDa. Observed band size: 38 kDa.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockout of EpCAM was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR701274_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of EpCAM was performed by loading 30 µg of A-431 wild type (Lane 1), A-431 Cas9 (Lane 2) andA-431 EpCAM KO (Lane 3) membrane enriched extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-EpCAM Recombinant Rabbit Monoclonal Antibody (28) (Product # MA5-29246, 1:500 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:10000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to EpCAM.

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence staining of Human EpCAM in SKBR3 cells. Cells were fixed with 4% PFA, blocked with 10% serum, and incubated with EpCAM Recombinant Rabbit Monoclonal Antibody (28) (Product # MA5-29246, 1:500) at 4°C overnight. Then cells were stained with the Alexa Fluor® 488-conjugated Goat Anti-rabbit IgG secondary antibody (green) and counterstained with DAPI (blue). Positive staining was localized to plasma membrane.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence staining of Human EpCAM in SKBR3 cells. Cells were fixed with 4% PFA, blocked with 10% serum, and incubated with EpCAM Recombinant Rabbit Monoclonal Antibody (28) (Product # MA5-29246, 1:500) at 4°C overnight. Then cells were stained with the Alexa Fluor® 488-conjugated Goat Anti-rabbit IgG secondary antibody (green) and counterstained with DAPI (blue). Positive staining was localized to plasma membrane.

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemical staining of human EpCAM in human bladder carcinoma with EpCAM Recombinant Rabbit Monoclonal Antibody (28) (Product # MA5-29246, 1:2,500 dilution, formalin-fixed paraffin embedded sections). Positive staining was localized to membrane of transitional epithelium.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemical staining of human EpCAM in human colon carcinoma with EpCAM Recombinant Rabbit Monoclonal Antibody (28) (Product # MA5-29246, 1:2,500 dilution, formalin-fixed paraffin embedded sections). Positive staining was localized to membrane of colonic gland epithelium.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow cytometric analysis of anti-EpCAM (CD326) reactivity on SKBR3 cells. SKBR3 cells were stained with EpCAM Monoclonal Antibody (Product # MA5-29246), then a FITC-conjugated second step antibody. The histogram were derived from the gated events based on light scattering characteristics of viable cells.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow cytometric analysis of EpCAM (CD326) in SKBR3 cells. SKBR3 cells were stained with EpCAM Recombinant Rabbit Monoclonal Antibody (28) (Product # MA5-29246), then a FITC-conjugated Secondary antibody. The histogram were derived from the gated events based on light scattering characteristics of viable cells.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

EpCAM Immunoprecipitation using: Lane A: 0.5 mg MCF-7 Whole Cell Lysate, Lane B: 0.5 mg A431 Whole Cell Lysate, Lane C: 0.5 mg HepG2 Whole Cell Lysate, Lane D: 0.5 mg Caco-2 Whole Cell Lysate 0.5 µL with EpCAM Recombinant Rabbit Monoclonal Antibody (28) (Product # MA5-29246) and 15 µL of 50 % Protein G agarose. Primary antibody: EpCAM Recombinant Rabbit Monoclonal Antibody (28), at 1:5,000 dilution. Secondary antibody: Dylight 800-labeled antibody to rabbit IgG (H+L), at 1:5,000 dilution. Developed using the Odyssey technique. Performed under reducing conditions. Predicted band size: 40 kDa. Observed band size: 40 kDa.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Isolation of CTC clusters from ovarian and prostate cancer patients. a Fluorescence microscope images of the isolated CTC clusters from ovarian cancer patients. Captured cells were stained with Cytokeratin, Vimentin (green), CD45 (red), and DAPI (nuclei, blue). Scale bars, 20 mum. b Images of the isolated CTC clusters from metastatic prostate cancer patients. Captured cells were stained with Cytokeratin, Vimentin, PSA/KLK3, EpCAM (green), CD45(red), and DAPI (nuclei, blue). Scale bars, 20 mum. c Clusters isolated from the peripheral blood and ascites sample drawn from the peritoneal cavity of an ovarian cancer patient at the same timepoint. The isolated CTC cluster was morphologically different compared to the tumor spheroids isolated from ascites sample of the same patient. The spheroids from ascites sample were fixed and stained with Cytokeratin, EpCAM (red), CD45 (green) and DAPI (nuclei, blue), and CTC clusters isolated from blood sample were fixed and stained with Cytokeratin, Vimentin (green), CD45 (red) and DAPI (nuclei, blue). Scale bars, 20 mum. d Percentage of prostate ( n = 8) and ovarian ( n = 9) cancer patients that are observed to have CTC cluster(s) in their blood samples. e Number of CTC clusters observed per milliliter of blood samples from prostate and ovarian cancer patients. f Distribution of the number of cells observed in the prostate and ovarian CTC clusters. The box plots show the 25th and 75th percentiles, line shows median, and whiskers show maxima a