Alexa Fluor® 647 anti-human CD326 (EpCAM) Antibody

Product Details

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.

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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)

1. Lammers R, et al. 2002. Exp. Hematol. 30:537.
2. Schultz LD, et al. 2010. P. Natl. Acad. Sci. USA 107:13022. PubMed
3. Human Protein Atlas http://www.proteinatlas.org/ENSG00000119888/antibody (IHC)
4. Radtke AJ, et al. 2020. Proc Natl Acad Sci USA. 117:33455-33465. (SB) PubMed
5. Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed

Product Citations

1. Liang J, et al. 2022. J Clin Invest. 132: . PubMed
2. Martínez-Sabadell A, et al. 2022. Cell Rep. 41:111430. PubMed
3. Estrada HQ, et al. 2022. Inflamm Bowel Dis. 28:667. PubMed
4. Bhatia S, et al. 2022. Cancer Res. 82:1174. PubMed
5. Sun L, et al. 2021. Cancer Cell. 39:1361. PubMed
6. Qin XY, et al. 2023. Cell Death Dis. 14:358. PubMed
7. Meirelles K, et al. 2012. Proc Natl Acad Sci U S A. 109:2358. PubMed
8. Pham K, et al. 2016. Am J Pathol. 186: 1537-1546. PubMed
9. Qin XY, et al. 2020. Cell Death Dis. 0.504166667. PubMed
10. Somerville TD, et al. 2020. eLife. 9:e53381.. PubMed
11. Hendricks A, et al. 2021. Front Oncol. 11:646885. PubMed
12. Ebisudani T, et al. 2021. Cell Reports. 35(10):109218. PubMed
13. Kitajima S, et al. 2019. Cancer Discov. 9:34. PubMed
14. Robertson N, et al. 2020. J Biol Chem. . PubMed
15. Campisi M, et al. 2020. Front Immunol. 1.909722222. PubMed
16. Csizmar CM, et al. 2019. J Am Chem Soc. 141:251. PubMed
17. Hendricks A, et al. 2020. Cancers (Basel). 12:00. PubMed
18. Rosenbluth JM, et al. 2020. Nat Commun. 11:1711. PubMed
19. Liu T, et al. 2019. Oncol Lett. 18:2262. PubMed
20. Stallcop LE, et al. 2018. Lab Chip. 18:451. PubMed
21. Arenas EJ, et al. 2021. Nat Commun. 12:1237. PubMed
22. Vanslambrouck JM, et al. 2022. Nat Commun. 13:5943. PubMed
23. Zhou J, et al. 2011. J Vis Exp. 55: 3322. PubMed
24. Robertson N, et al. 2020. J Biol Chem. 295:18436. PubMed
25. Liu X, et al. 2021. iScience. 24(6):102551. PubMed
26. Freire‐Pritchett P et al. 2017. eLife. 6 pii: e21926. PubMed
27. Sun Q, et al. 2018. J Clin Invest. 128:531. PubMed
28. Morris EJ, et al. 2020. Cell Rep. 30:3605. PubMed

RRID

AB_756086 (BioLegend Cat. No. 324212)

Antigen Details

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 View all products for this Gene ID

UniProt

View information about CD326 on UniProt.org

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Protocols

• Cell Surface Flow Cytometry Staining Protocol

Related Pages & Pathways

Related FAQs

If an antibody clone has been previously successfully used in IBEX in one fluorescent format, will other antibody formats work as well?

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.

Will antibodies my lab is already using for fluorescent or chromogenic IHC work in IBEX?

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.

Are other fluorophores compatible with IBEX?

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.

The same antibody works in one tissue type but not another. What is happening?

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.

How can I be sure the staining I’m seeing in my tissue is real?

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.

Other Formats

Certificate of Analysis