PerCP/Cyanine5.5 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 PerCP/Cyanine5.5 under optimal conditions.

Concentration

Lot-specific (to obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.)

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
SB - Community Verified

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.

* PerCP/Cyanine5.5 has a maximum absorption of 482 nm and a maximum emission of 690 nm.

Excitation Laser

Blue Laser (488 nm)

Application Notes

Additional reported applications (for the revelant formats) include: immunofluorescence, immunohistochemistry³, and spatial biology (IBEX)^4,5.

Additional Product Notes

For the use of this antibody in spatial biology (SB), we have partnered with Bruker Spatial Biology Biosciences for demonstration of this antibody on their next-generation ChipCytometry instrument called the CellScape™. The CellScape platform is an end-to-end solution for highly multiplexed spatial omics. Combining an advanced, purpose-built imaging system with easy-to-use fluidics for walk-away automation, the CellScape system will accelerate your exploration into the rapidly evolving field of spatial biology. More information on the the Bruker Spatial Biology CellScape and a complete list of our antibodies that have been demonstrated on the instrument can be found here.

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. Lao L, et al. 2023. Cancer Immunol Res. 11:320. PubMed
2. Ge W, et al. 2022. Int J Mol Sci. 24: . PubMed
3. Otano I, et al. 2021. Nat Commun. 12:7296. PubMed
4. Cheng L, et al. 2021. Cancer Immunol Immunother. Online ahead of print. PubMed
5. Gui J, et al. 2011. Int Immunol. 1.407638889. PubMed
6. Willingham S, et al. 2012. Proc Natl Acad Sci U S A. 109:6627. PubMed
7. Wang J, et al. 2020. Cell. 183:1264. PubMed
8. Cai X, et al. 2014. PLoS One. 9:108942. PubMed
9. Costa A et al. 2018. Cancer cell. 33(3):463-479 . PubMed
10. Cheah M, et al. 2015. Proc Natl Acad Sci U S A. 112:4725. PubMed
11. Lau KX, et al. 2020. Nat Commun. 11:2420. PubMed
12. Uniken Venema WT, et al. 2019. Gastroenterology. 156:812. PubMed
13. Fridriksdottir A, et al. 2015. Nat Commun. 6: 8786. PubMed
14. Montel‐Hagen A et al. 2019. Cell stem cell. 24(3):376-389 . PubMed
15. Kim J, et al. 2018. J Histochem Cytochem. 66:879. PubMed

RRID

AB_2098808 (BioLegend Cat. No. 324213)
AB_2098808 (BioLegend Cat. No. 324214)

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

How stable is PerCP/Cyanine5.5 tandem as compared to PerCP alone?

PerCP/Cyanine5.5 is quite photostable and also better than PerCP alone in withstanding fixation.

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