Left: Intracellular staining of fixed and permeabilized F9 cells with 0.03 µg of Rat IgG2a κ Isotype Control Alexa Fluor 647 (open histogram) or 0.03 µg of Anti-Mouse Nanog Alexa Fluor® 647 (filled histogram). Right: Intracellular staining of fixed and permeabilized F9 cells cultured for 3 days with 1 µM All-Trans-Retinoic Acid (ATRA) with 0.03 µg Rat IgG2a κ Isotype Control Alexa Fluor 647 (open histogram) or 0.03 µg of Mouse Nanog Alexa Fluor® 647 (filled histogram). All intracellular staining was performed using the Foxp3 Staining Buffer Set (cat. 00-5523).
Contents: Anti-Mouse Nanog Alexa Fluor® 647 Catalog Number: 51-5761 Concentration: 0.2 mg/ml Formulation: Phosphate buffered saline, pH 7.2, less than or equal to 0.09% NaN3, BSA and/or gelatin for protein stabilization Storage Conditions: Store at 2-8°C. Do not freeze. Light sensitive material. Clone: eBioMLC-51 Host/Isotype: Rat IgG2a
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51-5761-80
25 ug
Note: Several countries will continue to be supplied via distributors. Country specific prices may apply.
Flow Cytometry Product Notes: Test Sizes: To accommodate multicolor flow cytometry, eBioscience is in the process of reducing test size volumes from 20 µl to 5 µl. Please check your antibody vial for the recommended test size. Fluorochrome Replacements: eBioscience is in the process of replacing all Alexa Fluor® 647 conjugated products with eFluor® 660 conjugated products.
Description
The eBioMLC51 monoclonal antibody recognizes mouse Nanog. Nanog is a multidomain homeobox transcription factor that has been shown to maintain pluripotency of embryonic stem cells, independent of LIF/Stat3. Expression of Nanog in the mouse is specific to early embryos, the ICM of the blastocyst, embryonic stem (ES) cells, and embryonic germ (EG) cells. Nanog expression often overlaps, but is not identical to, that of Oct4. Nanog is downregulated upon cellular differentiation and loss of pluripotency, making it a suitable marker in determining the undifferentiated state of stem cells.
Nanog acts as a transcriptional activator and has two activation domains in the C-terminus, called CD2 and WR, and one activation domain in the N terminus. The CD2 domain is unique to Nanog, whereas the NK2 DNA binding domain is well conserved.
Immunoblotting using eBioMLC51 reveals a band at ~ 45 kDa in F9 (an embryonal carcinoma cell line) lysate, but not in lysate from the NIH3T3 cell line or mouse spleen.
Preliminary data using fluorochrome-conjugated eBioMLC51 suggests that it is essential to use the eBioscience Foxp3 Staining Buffer Set, Cat. 00-5523, for intracellular staining of mouse Nanog.
Applications Reported
For research use only, not for diagnostic or therapeutic use. This eBioMLC-51 antibody has been reported for use in intracellular staining followed by flow cytometric analysis.
Applications Tested
This eBioMLC-51 antibody has been tested by intracellular staining followed by flow cytometric analysis of the F9 cell line. This can be used at less than or equal to 0.03 μg 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 105 to 108 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.
Important: eBioscience Foxp3 Staining Buffer Set, Cat. 00-5523, must be used for fixation and permeablization followed by intracellular staining with eBioMLC51.
References
Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Maruyama, M., Maeda, M., Yamanaka, S. 2003. The Homeoprotein Nanog is Required for Maintenance of Pluripotency in Mouse Epiblast and ES Cells. Cell 113:631-642
Yanmei Chen, Zhongwei Du, Zhen Yao. 2006. Roles of the Nanog Protein in Murine F9 Embryonal Carcinoma Cells and Their Endoderm-Differentiated Counterparts. Cell Research 16:641-650.
Guangjin Pan and Duanqing Pei. 2005. The Stem Cell Pluripotency Factor NANOG Activates Transcription with Two Unuasually Potent Subdomains at Its C Terminus. The Journal of Biological Chemistry Vol.280, No.2, 1401-1407.
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Alexa Fluor® and Pacific Blue® are registered trademarks of and licensed under patents assigned to Molecular Probes, Inc. for research use only. This product is subject to an agreement between Molecular Probes, Inc. and eBioscience, and the manufacture, use, sale or import of this product may be subject to one or more U.S. patents, pending applications and corresponding foreign equivalents, owned by Molecular Probes, Inc. (a wholly owned subsidiary of Invitrogen Corp). The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product for life science research or as an ASR. The buyer cannot use this product for manufacturing or for any other screening (specifically including use in combination with microarrays or High Content Screening) or testing purpose, other than as an ASR. For information on purchasing a license to this product for purposes other than life science research or use as an ASR, contact Molecular Probes, Inc.
