Biolegend, 147307, Alexa Fluor® 647 anti-mouse/human CD324 (E-Cadherin) Antibody, 25μg

CD324, also known as E-cadherin, cadherin-1, CDH1, and UVO is a member of the cadherin superfamily. It is a calcium-dependent, transmembrane cell-cell adhesion glycoprotein composed of four extracellular cadherin repeats and a highly conserved cytoplasmic tail region. CD324 is widely expressed in epithelial cells in the colon, uterus, liver, keratinocytes, brain, heart, muscle, kidney, and pancreas as well as erythroid cells. CD324 functions as a cell adhesion molecule involved in development, bacterial pathogenesis, and tumor invasion. In bacterial pathogenesis, the ectodomain of CD324 mediates bacterial adhesion to mammalian cells, while the cytoplasmic domain is required for internalization. CD324 binds to the αEβ7 integrin to mediate cell adhesion and also interacts with a number of intracellular proteins including including erbin, ezrin, caspase-3, caspase-8, β-catenin, presenilin 1, and casein kinase II as well as other extracellular proteins including the EGF receptor.
25μg
Verified Reactivity: Mouse, Human
Reported Reactivity: Cynomolgus, Dog, Pig
Antibody Type: Monoclonal
Host Species: Rat
Immunogen: E-Cadherin extracellular domain
Formulation: Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide.
Preparation: The antibody was purified by affinity chromatography and conjugated with Alexa Fluor® 647 under optimal conditions.
Concentration: 0.5 mg/mL
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, IHC-F, 3D IHC - 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 ≤ 1.0 µg per million cells in 100 µL volume. For immunocytochemistry, a concentration range of 2.0 - 10 μg/mL is recommended. For immunohistochemical staining on frozen tissue sections, a concentration range of 5.0 - 10 µg/mL is suggested. For 3D immunohistochemistry on formalin-fixed tissues, a concentration of 5.0 µg/mL is suggested. It is recommended that the reagent be titrated for optimal performance for each application. It is also recommended using EDTA-based solutions for dissociating attachment-dependent cell lines. * Alexa Fluor® 647 has a maximum emission of 668 nm when it is excited at 633 nm / 635 nm. 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 relevant formats) include: immunoprecipitation1, Western Blotting1, immunomicroscopy3, biological function1,2, 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): Vestweber D, et al. 1985. EMBO. 4:3393. (IP, WB, FA) Nakagawa M, et al. 2001. J. Cell Sci. 114:1829. (FA in canine cells) Mohamet L, et al. 2010. PLoS ONE. 5:e12921. (IF) Radtke AJ, et al. 2020. Proc Natl Acad Sci U S A. 117:33455-65. (SB) PubMed Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed
Product Citations: Jeon Y, et al. 2022. Cancers (Basel). 14:. PubMed Vereertbrugghen A, et al. 2023. J Neuroinflammation. 20:120. PubMed Smolen JA, et al. 2022. PNAS Nexus. 1:pgac235. PubMed Khodeer S, et al. 2022. J Cell Sci. 135: . PubMed Law NC, et al. 2019. Nat Commun. 10:2787. PubMed Rodríguez E, et al. 2022. Commun Biol. 5:41. PubMed Cohen SB et al. 2018. Cell host & microbe. 24(3):439-446 . PubMed Pitarresi JR, et al. 2021. Cancer Discov. 11:1774. PubMed Stefkovich ML, et al. 2021. Curr Protoc. 1:e139. PubMed Gentek R, et al. 2018. Immunity. 48:1160. PubMed Han S, et al. 2019. Cell Stem Cell. 25:342. PubMed Rosàs-Canyelles E, et al. 2020. Sci Adv. 6:eaay1751. PubMed Emily A Thompson et al. 2019. Cell reports. 26(11):2859-2867 . PubMed Steele NG, et al. 2021. Clin Cancer Res. 27:2023. PubMed Gawronska-Kozak B, et al. 2016. PLoS One. 11: 0150635. PubMed Norgard RJ, et al. 2021. EMBO Rep. 22:e51872. PubMed Malenica I, et al. 2021. Nat Commun. 12:5209. PubMed Radke J, et al. 2022. Nat Commun. 13:7304. PubMed
RRID: AB_2563954 (BioLegend Cat. No. 147307) AB_2563955 (BioLegend Cat. No. 147308)
Structure: Member of the cadherin superfamily. Calcium-dependent, transmembrane cell-cell adhesion glycoprotein composed of four extracellular cadherin repeats and a highly conserved cytoplasmic tail region.
Distribution: Widely expressed in epithelial cells in the colon, uterus, liver, keratinocytes, brain, heart, muscle, kidney, and pancreas as well as erythroid cells.
Function: Cell adhesion molecule involved in development, bacterial pathogenesis, and tumor invasion. The ectodomain of CD324 mediates bacterial adhesion to mammalian cells, while the cytoplasmic domain is required for internalization.
Interaction: Interacts with a variety of proteins including erbin, ezrin, caspase-3, caspase-8, EGF receptor, β-catenin, presenilin 1, casein kinase II, and others.
Ligand/Receptor: αEβ7 integrin.
Cell Type: Embryonic Stem Cells
Biology Area: Cell Adhesion, Cell Biology, Immunology, Innate Immunity, Neuroscience, Stem Cells, Synaptic Biology
Molecular Family: Adhesion Molecules, CD Molecules
Antigen References: 1. Overduin M, et al. 1995. Science 267:386. 2. Boggon TJ, et al. 2002. Science 296:1308. 3. Berx G, et al. 1995. EMBO J. 14:6107. 4. Perl AK, et al. 1998. Nature 392:190.
Gene ID: 99912550
UniProt: View information about CD324 on UniProt.org
Clone: DECMA-1
Regulatory Status: RUO
Other Names: E-Cadherin, Cadherin-1, CDH1, and UVO
Isotype: Rat IgG1, 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.