Biolegend, 324211, Alexa Fluor® 647 anti-human CD326 (EpCAM) Antibody, 25tests

CD326 is also known as Ep-CAM, tumor associated calcium signal transducer 1, epithelial cell surface antigen, epithelial glycoprotein 2, EGP2, adenocarcinoma associated antigen, and TROP1. CD326 is a type I transmembrane protein containing six disulfide bridges and one THYRO domain. This cell surface glycosylated 40 kD protein is highly expressed in bone marrow, colon, lung, and most normal epithelial cells and is expressed on carcinomas of gastrointestinal origin. Recently, it has been reported that CD326 expression occurs during the early steps of erythrogenesis. CD326 functions as a homotypic calcium-independent cell adhesion molecule and is believed to be involved in carcinogenesis by its ability to induce genes involved in cellular metabolism and proliferation. CD326 antigen is an immunotherapeutic target for the treatment of human carcinomas.
25tests
Verified Reactivity: Human
Antibody Type: Monoclonal
Host Species: Mouse
Immunogen: DU.4475 breast carcinoma
Formulation: Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide and BSA (origin USA)
Preparation: The antibody was purified by affinity chromatography and conjugated with Alexa Fluor® 647 under optimal conditions.
Concentration: Lot-specific (to obtain lot-specific concentration, please enter the lot number in our Concentration and Expiration Lookup or Certificate of Analysis online tools.)
Storage & Handling: The antibody solution should be stored undiluted between 2°C and 8°C, and protected from prolonged exposure to light. Do not freeze.
Application: FC - Quality tested ICC - Verified SB - Reported in the literature, not verified in house
Recommended Usage: Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 5 µL per million cells in 100 µL staining volume or 5 µL per 100 µL of whole blood. It is recommended that the reagent be titrated for optimal performance for each application. * Alexa Fluor® 647 has a maximum emission of 668 nm when it is excited at 633nm / 635nm. Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.View full statement regarding label licenses
Excitation Laser: Red Laser (633 nm)
Application Notes: Additional reported applications (for the revelant formats) include: immunofluorescence, immunohistochemistry3, and spatial biology (IBEX)4,5.
Additional Product Notes: Iterative Bleaching Extended multi-pleXity (IBEX) is a fluorescent imaging technique capable of highly-multiplexed spatial analysis. The method relies on cyclical bleaching of panels of fluorescent antibodies in order to image and analyze many markers over multiple cycles of staining, imaging, and, bleaching. It is a community-developed open-access method developed by the Center for Advanced Tissue Imaging (CAT-I) in the National Institute of Allergy and Infectious Diseases (NIAID, NIH).
Application References(PubMed link indicates BioLegend citation): Lammers R, et al. 2002. Exp. Hematol. 30:537. Schultz LD, et al. 2010. P. Natl. Acad. Sci. USA 107:13022. PubMed Human Protein Atlas http://www.proteinatlas.org/ENSG00000119888/antibody (IHC) Radtke AJ, et al. 2020. Proc Natl Acad Sci USA. 117:33455-33465. (SB) PubMed Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed
Product Citations: Sun L, et al. 2021. Cancer Cell. 39:1361. PubMed Qin XY, et al. 2023. Cell Death Dis. 14:358. PubMed Liang J, et al. 2022. J Clin Invest. 132: . PubMed Martínez-Sabadell A, et al. 2022. Cell Rep. 41:111430. PubMed Estrada HQ, et al. 2022. Inflamm Bowel Dis. 28:667. PubMed Bhatia S, et al. 2022. Cancer Res. 82:1174. PubMed Meirelles K, et al. 2012. Proc Natl Acad Sci U S A. 109:2358. PubMed Pham K, et al. 2016. Am J Pathol. 186: 1537-1546. PubMed Qin XY, et al. 2020. Cell Death Dis. 0.504166667. PubMed Somerville TD, et al. 2020. eLife. 9:e53381.. PubMed Hendricks A, et al. 2021. Front Oncol. 11:646885. PubMed Ebisudani T, et al. 2021. Cell Reports. 35(10):109218. PubMed Kitajima S, et al. 2019. Cancer Discov. 9:34. PubMed Robertson N, et al. 2020. J Biol Chem. . PubMed Campisi M, et al. 2020. Front Immunol. 1.909722222. PubMed Csizmar CM, et al. 2019. J Am Chem Soc. 141:251. PubMed Hendricks A, et al. 2020. Cancers (Basel). 12:00. PubMed Rosenbluth JM, et al. 2020. Nat Commun. 11:1711. PubMed Liu T, et al. 2019. Oncol Lett. 18:2262. PubMed Stallcop LE, et al. 2018. Lab Chip. 18:451. PubMed Arenas EJ, et al. 2021. Nat Commun. 12:1237. PubMed Vanslambrouck JM, et al. 2022. Nat Commun. 13:5943. PubMed Zhou J, et al. 2011. J Vis Exp. 55: 3322. PubMed Liu X, et al. 2021. iScience. 24(6):102551. PubMed Freire‐Pritchett P et al. 2017. eLife. 6 pii: e21926. PubMed Sun Q, et al. 2018. J Clin Invest. 128:531. PubMed Morris EJ, et al. 2020. Cell Rep. 30:3605. PubMed Robertson N, et al. 2020. J Biol Chem. 295:18436. PubMed
RRID: AB_756086 (BioLegend Cat. No. 324212)
Structure: Type I transmembrane protein, contains six disulfide bridges, one THYRO domain, approximate molecular weight 40 kD.
Distribution: Highly expressed in bone marrow, colon, lung, and most normal epithelial cells. Also highly expressed on carcinomas of gastrointestinal origin. Expressed during early erythrogenesis.
Function: Homotypic calcium-independent cell adhesion. CD326 is believed to be involved in carcinogenesis by its ability to induce genes involved in cellular metabolism and proliferation.
Modification: Glycosylated.
Cell Type: Embryonic Stem Cells, Epithelial cells
Biology Area: Cell Biology, Immunology, Stem Cells
Molecular Family: Adhesion Molecules, CD Molecules
Antigen References: 1. Strnad J, et al. 1989. Cancer Res. 49:314. 2. Munz M, et al. 2004. Oncogene 23:5748. 3. Rao CG, et al. 2005. Int. J. Oncol. 27:49.
Gene ID: 4072
UniProt: View information about CD326 on UniProt.org
Clone: 9C4
Regulatory Status: RUO
Other Names: Ep-CAM, tumor associated calcium signal transducer 1, epithelial cell surface antigen, epithelial glycoprotein 2, EGP2, adenocarcinoma associated antigen, TROP1
Isotype: Mouse IgG2b, κ
Q: If an antibody clone has been previously successfully used in IBEX in one fluorescent format, will other antibody formats work as well?
A: It’s likely that other fluorophore conjugates to the same antibody clone will also be compatible with IBEX using the same sample fixation procedure. Ultimately a directly conjugated antibody’s utility in fluorescent imaging and IBEX may be specific to the sample and microscope being used in the experiment. Some antibody clone conjugates may perform better than others due to performance differences in non-specific binding, fluorophore brightness, and other biochemical properties unique to that conjugate.
Q: Will antibodies my lab is already using for fluorescent or chromogenic IHC work in IBEX?
A: Fundamentally, IBEX as a technique that works much in the same way as single antibody panels or single marker IF/IHC. If you’re already successfully using an antibody clone on a sample of interest, it is likely that clone will have utility in IBEX. It is expected some optimization and testing of different antibody fluorophore conjugates will be required to find a suitable format; however, legacy microscopy techniques like chromogenic IHC on fixed or frozen tissue is an excellent place to start looking for useful antibodies.
Q: Are other fluorophores compatible with IBEX?
A: Over 18 fluorescent formats have been screened for use in IBEX, however, it is likely that other fluorophores are able to be rapidly bleached in IBEX. If a fluorophore format is already suitable for your imaging platform it can be tested for compatibility in IBEX.
Q: The same antibody works in one tissue type but not another. What is happening?
A: Differences in tissue properties may impact both the ability of an antibody to bind its target specifically and impact the ability of a specific fluorophore conjugate to overcome the background fluorescent signal in a given tissue. Secondary stains, as well as testing multiple fluorescent conjugates of the same clone, may help to troubleshoot challenging targets or tissues. Using a reference control tissue may also give confidence in the specificity of your staining.
Q: How can I be sure the staining I’m seeing in my tissue is real?
A: In general, best practices for validating an antibody in traditional chromogenic or fluorescent IHC are applicable to IBEX. Please reference the Nature Methods review on antibody based multiplexed imaging for resources on validating antibodies for IBEX.