Invitrogen™ CD326 (EpCAM) Monoclonal Antibody (G8.8), Super Bright™ 645, eBioscience™, Invitrogen™

Catalog No.	Quantity
64-579-182	100 μg

Catalog No. 64-579-182 Supplier Invitrogen™ Supplier No. 64579182

Description

Rat Monoclonal Antibody

Description: The eBioC1.7 monoclonal antibody reacts with human CD244 (2B4, p38). In human, CD244 is a 38 kDa protein expressed on NK cells, a subset of CD8+ T cells, gammadelta T cells, monocytes, basophils and eosinophils. Binding of the CD244 ligand, CD48, results in NK cell activation, unlike mouse CD244, which is an inhibitory receptor. For CD244 expressed on NK cells, binding of CD48 results in enhanced NK cell cytotoxicity and secretion of IFN-gamma. Recently, it was demonstrated that binding of the C1.7 monoclonal antibody and CD48 involve the same residue in the V domain of human CD244, which explains the ability of C1.7 binding to induce activation of NK cells. Binding of C1.7 to CD244 leads to tyrosine phosphorylation and recruitment of the adaptor molecule SAP (SLAM-associated protein). Patients with X-linked lymphoproliferative disorder (XLPD) have a mutation in SAP which renders it unable to bind to phosphorylated CD244. Applications Reported: This eBioC1.7 antibody has been reported for use in flow cytometric analysis. Applications Tested: This eBioC1.7 antibody has been pre-diluted and tested by flow cytometric analysis of normal human peripheral blood cells. This may be used at 5 μL (1 μg/mL) per test. A test is defined as the amount (μg) of antibody that will stain a cell sample in a final volume of 100 μL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test.

Ep-CAM (epithelial adhesion molecule, epithelial specific antigen, ESA) is a transmembrane glycoprotein expressed in the epithelium with a molecular weight of approximately 40 kDa, which functions as an epithelial cell adhesion molecule. Ep-CAM functions as a homotypic calcium-independent cell adhesion molecule, and has a direct impact on cell cycle, proliferation and metabolism of epithelial cells and fibroblasts due to its ability to rapidly induce the proto-oncogene c-myc and the cell cycle regulating genes cyclin A and E. Ep-CAM mediates Ca2+-independent homotypic interactions. Formation of Ep-CAM-mediated adhesions have a negative regulatory effect on adhesions mediated by classic cadherins, which may have strong effects on the differentiation and growth of epithelial cells. Ep-CAM overexpression was suggested to be associated with enhanced epithelial proliferation. Ep-CAM is highly expressed in human carcinomas, and is a marker for tumors of epithelial lineage. Ep-CAM is expressed on baso-lateral cell surface in most simple epithelia and many carcinoma types. Also, Ep-CAM reportedly distinguishes adenocarcinomas from pleural mesotheliomas.

Specifications

• Antigen: CD326 (EpCAM)
• Applications: Flow Cytometry
• Classification: Monoclonal
• Clone: G8.8
• Concentration: 0.2 mg/mL
• Conjugate: Super Bright 645
• Formulation: PBS with BSA and 0.09% sodium azide; pH 7.2
• Gene: EPCAM
• Gene Accession No.: Q99JW5
• Gene Alias: adenocarcinoma-associated antigen; CD326; cell surface glycoprotein Trop-1; DIAR5; EGP; EGP-2; EGP314; EGP40; EPCAM; Ep-CAM; EpCAM1; epithelial cell adhesion molecule; Epithelial cell surface antigen; Epithelial glycoprotein; Epithelial glycoprotein 314; ESA; GA733-2; gp40; hEGP314; HNPCC8; human epithelial glycoprotein-2; KS 1/4 antigen; KS1/4; KSA; Ly74; lymphocyte antigen 74; M1S2; M4S1; major gastrointestinal tumor-associated protein GA733-2; mEGP314; membrane component, chromosome 4, surface marker (35kD glycoprotein); MIC18; MK-1; panepithelial glycoprotein 314; protein 289A; Protein D5.7A; Tacsd1; Tacstd1; TROP1; Trop-1 protein; Tumor-associated calcium signal transducer 1
• Gene Symbols: EPCAM
• Host Species: Rat
• Purification Method: Affinity chromatography
• Quantity: 100 μg
• Regulatory Status: RUO
• Primary or Secondary: Primary
• Gene ID (Entrez): 17075
• Target Species: Mouse
• Content And Storage: 4°C, store in dark, DO NOT FREEZE!
• Product Type: Antibody
• Form: Liquid
• Isotype: IgG2a κ

Frequently Asked Questions (FAQs)

Can I use the OneComp and UltraComp eBeads microspheres with Super Bright-conjugated antibodies?

UltraComp eBeads microspheres (Cat. No. 01-2222) are recommended for use with Super Bright dyes.
Note: Super Bright Staining Buffer (Cat. No. SB-4400) is not compatible with UltraComp eBeads microspheres (Cat. No. 01-2222-41, 00-2222-42). If using UltraComp eBeads microspheres as a compensation tool, solely use Flow Cytometry Stain Buffer (Cat. No. 00-4222-26, 00-4222-57) for any antibody dilutions.

In some experiments, we have observed that compensation values for Super Bright 780- and Brilliant Violet 785- or Brilliant Violet 786-conjugated antibodies are higher in the violet 450/50 channel when using UltraComp eBeads microspheres as compared to single-color stained cells. In such circumstances, we would recommend setting compensation with cells. We have also observed this in some experiments using AbC Total Antibody Compensation beads, both with Super Bright 780 and Brilliant Violet 786. We have not tested Brilliant Violet 785 with the AbC beads.

Can I prepare an antibody cocktail containing Super Bright Staining Buffer and Super Bright-conjugated antibodies ahead of time?

We recommend that the antibody cocktails containing Super Bright-conjugated antibodies and Super Bright Staining Buffer are prepared fresh prior to staining. Discard any unused portions. We do not recommend overnight storage of prepared cocktails.

Can I fix my cells after staining with Super Bright-conjugated antibodies? How long can fixed cells be stored prior to analysis?

Samples that have been stained with antibodies conjugated to Super Bright dyes may be stored for up to three days, at 2-8°C, in the dark, using either IC Fixation Buffer (Cat. No. 00-8222) or 1-step Fix/Lyse Buffer (Cat. No. 00-5333) with no significant effect on brightness or compensation.

Are the Super Bright dyes sensitive to methanol fixation?

Super Bright dyes are stable in methanol-based fixation buffers.

Can Super Bright-conjugated antibodies be used in combination with intracellular (IC Fixation/Permeabilization) or intranuclear (Foxp3 fixation/permeabilization) staining?

Yes, Super Bright-conjugated antibodies are stable in formaldehyde-based fixation buffers and permeabilization buffers, such as the IC Fixation and Permeabilization Buffer Set (Cat. No. 88-8824) and the Foxp3/Transcription Factor Staining Buffer Set (Cat. No. 00-5523).

Which laser do I use to detect Super Bright-conjugated antibodies?

The violet laser (405 nm) should be used to excite Super Bright-conjugated antibodies.

Can the Super Bright Staining Buffer be used with other polymer dyes?

Yes, the Super Bright Staining Buffer (Cat. No. SB-4400) is compatible with other polymer dyes (i.e., Brilliant Violet dyes) and is useful for minimizing any non-specific polymer interactions when two or more of these dyes are used in combination.

Can Super Bright-conjugated antibodies be used in combination with other polymer dyes? What buffer should I use when using more than one Super Bright or polymer dye?

When using two or more Super Bright dyes, or when using Super Bright dyes in combination with other polymer dyes (i.e., Brilliant Violet dyes), we recommend using the Super Bright Staining Buffer (Cat. No. SB-4400) to minimize any non-specific polymer interactions between these fluorochromes. For specific instructions for use, please refer to the product technical data sheet.

Are there recommended tubes for staining with Super Bright-conjugated antibodies?

We recommend using polystyrene tubes (for example, FACS tubes) for staining with your Super Bright-conjugated antibodies. If using polypropylene (for example, Eppendorf tubes), protecting from light is critical.

Is there a specific buffer I should use when staining with Super Bright-conjugated antibodies?

No special buffer is required when using a single Super Bright-conjugated antibody in a panel.
When using more than one Super Bright dye, or when using Super Bright dyes in combination with other polymer dyes (i.e., Brilliant Violet dyes), we recommend using the Super Bright Staining Buffer (Cat. No. SB-4400) to minimize any non-specific polymer interactions between these fluorochromes. For specific instructions for use, please refer to the product technical data sheet.

How does the protocol for staining with Super Bright-conjugated antibodies differ from staining with conventional organic fluorochrome and eVolve conjugated antibodies?

Antibodies conjugated to Super Bright dyes can be used similarly to traditional fluorochromes. If multiple Super Bright dyes are used in combination with each other or in combination with other polymer dyes (i.e., Brilliant Violet dyes), then the use of Super Bright Staining Buffer (Cat. No. SB-4400) is recommended to minimize any non-specific polymer interactions between these fluorochromes. For specific instructions for use, please refer to the product technical data sheet.

Can the Super Bright dyes be used for non-flow applications?

Super Bright dyes have not been tested for applications other than flow cytometry.

What is the difference between the Super Bright and eVolve dyes?

Super Bright dyes are fluorochromes based on polymer technology and are excited by the violet laser. In contrast, eVolve dyes are based on Qdot nanocrystal technology and are maximally excited by the UV laser, although they may also be excited by violet, blue, yellow-green, and, depending on the eVolve, the red laser lines. The general shape of the emission spectra for Super Bright dyes will be more similar to traditional fluorochromes, while eVolve dyes have extremely narrow emission spectra. Please contact Tech Support (techsupport@thermofisher.com) for more information.

What are the Super Bright dyes?

eBioscience Super Bright dyes are a series of patent-pending fluorochromes that are based upon a fluorescent polymer and its tandems. Super Bright dyes have been developed for use in flow cytometry and can be excited by the violet laser (405 nm). The peak emission of each Super Bright dye is indicated by the number in the product description, e.g., "Super Bright 600" has a peak emission of 600 nm.

Are the Super Bright Dyes photo-labile?

As with other fluorochromes, we recommend minimal exposure to light to maintain optimal signal.