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Purified Mouse anti-Human Nanog
Purified Mouse anti-Human Nanog

Western Blot analysis of Nanog in human embryonic stem cell line. Lysate from H9 human ES cells* (WiCell, Madison, WI) was probed with Purified Mouse anti-Human Nanog monoclonal antibody at titrations of 2.0 (lane 1), 1.0 (lane 2), and 0.5 µg/ml (lane 3).  Nanog is identified as a band of 36-37 kDa.  

*The H9 cells were cultured on a mitomycin C-treated mouse embryonic fibroblast feeder layer [MEF (CF-1), ATCC SCRC-1040] that maintains the undifferentiated state of the ES cells.  The lysate was made from a mixture of the 2 cell types, the majority of which were H9 cells.

Purified Mouse anti-Human Nanog

Immunofluorescent staining of human ES cell line.  The H9 cell line was cultured, fixed, permeabilized, and stained with Purified Mouse anti-Human Nanog monoclonal antibody (pseudo-colored green) according to the Recommended Assay Procedure.  The second-step reagent was Alexa Fluor® 647 goat anti-mouse Ig (Invitrogen) and counter-staining was with Hoechst 33342 (pseudo-colored blue).  The images were captured on a BD Pathway™ 435 Cell Analyzer using a 10X objective and merged using BD Attovision™ software.  Saponin permeabilization is recommended for this antibody, but not Triton™ X-100 or cold methanol.

Purified Mouse anti-Human Nanog

Western Blot validation of Nanog by RNAi in human embryonal carcinoma cell line. Lysates from NTERA-2 cl.D1 cells (ATCC CRL-1973, lane 1) and Nanog RNAi-transfected NTERA-2 cl.D1 cells (lane 2) were probed with Purified Mouse anti-Human Nanog monoclonal antibody at 0.5 µg/ml.  Down-regulation of Nanog expression is evident in the RNAi-transfected cells (upper blot).  Purified Mouse anti-Actin monoclonal antibody (Cat. No. 612656 or 612657) was the gel-loading control (lower blot).  

Western Blot analysis of Nanog in human embryonic stem cell line. Lysate from H9 human ES cells* (WiCell, Madison, WI) was probed with Purified Mouse anti-Human Nanog monoclonal antibody at titrations of 2.0 (lane 1), 1.0 (lane 2), and 0.5 µg/ml (lane 3).  Nanog is identified as a band of 36-37 kDa.  

*The H9 cells were cultured on a mitomycin C-treated mouse embryonic fibroblast feeder layer [MEF (CF-1), ATCC SCRC-1040] that maintains the undifferentiated state of the ES cells.  The lysate was made from a mixture of the 2 cell types, the majority of which were H9 cells.

Immunofluorescent staining of human ES cell line.  The H9 cell line was cultured, fixed, permeabilized, and stained with Purified Mouse anti-Human Nanog monoclonal antibody (pseudo-colored green) according to the Recommended Assay Procedure.  The second-step reagent was Alexa Fluor® 647 goat anti-mouse Ig (Invitrogen) and counter-staining was with Hoechst 33342 (pseudo-colored blue).  The images were captured on a BD Pathway™ 435 Cell Analyzer using a 10X objective and merged using BD Attovision™ software.  Saponin permeabilization is recommended for this antibody, but not Triton™ X-100 or cold methanol.

Western Blot validation of Nanog by RNAi in human embryonal carcinoma cell line. Lysates from NTERA-2 cl.D1 cells (ATCC CRL-1973, lane 1) and Nanog RNAi-transfected NTERA-2 cl.D1 cells (lane 2) were probed with Purified Mouse anti-Human Nanog monoclonal antibody at 0.5 µg/ml.  Down-regulation of Nanog expression is evident in the RNAi-transfected cells (upper blot).  Purified Mouse anti-Actin monoclonal antibody (Cat. No. 612656 or 612657) was the gel-loading control (lower blot).  

Product Details
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BD Pharmingen™
Human (QC Testing)
Mouse BALB/c IgG1, κ
Human Nanog Recombinant Protein
Western blot (Routinely Tested), Bioimaging (Tested During Development)
36-37 kDa
0.5 mg/ml
AB_1645597
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

Recommended Assay Procedures

Bioimaging:

  1.        Seed the cells in appropriate culture medium at an appropriate cell density in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219), and

        culture overnight to 48 hours.

  2.        Remove the culture medium from the wells, wash the wells twice with 100 μl of 1× PBS, and fix the cells by adding 100 µl of fresh 3.7% Formaldehyde in PBS or BD Cytofix™ fixation buffer (Cat. No. 554655) to each well and incubating for 10 minutes at room temperature (RT).

  3.        Remove the fixative from the wells, and wash the wells twice with 100 μl of 1× PBS.

  4.        Permeabilize the cells by adding 100 µl of 1× BD Perm/Wash™ buffer (Cat. No. 554723) to each well and incubating for 30 minutes at RT.

  5.        Remove the permeabilizer, and wash the wells twice with 100 μl of 1× PBS.

  6.        Dilute the antibody in BD Perm/Wash™ buffer, and stain the cells by adding 50 µl of the diluted antibody to each well and incubating for 1 hour at RT.

  7.        Remove the diluted antibody, and wash the wells three times with 100 μl of 1× PBS.

  8.        Remove the PBS, dilute the second-step reagent in BD Perm/Wash™ buffer, and stain the cells by adding 50 µl of the diluted second-step reagent to each well and incubating for 1 hour at RT.

  9.        Remove the diluted second-step reagent, and wash the wells twice with 100 μl of 1× PBS.

10.        Remove the PBS, and counter-stain the nuclei by adding 100 μl of a 2 μg/ml solution of Hoechst 33342 (eg, Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.

11.        View and analyze the cells on an appropriate imaging instrument.

Bioimaging:  For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp

Western blot: For more detailed information please refer to http://www.bdbiosciences.com/pharmingen/protocols/Western_Blotting.shtml

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Triton is a trademark of the Dow Chemical Company.
  3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  4. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  5. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
560109 Rev. 2
Antibody Details
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L96-549

The L96-549 monoclonal antibody reacts with human Nanog (named for Tir Na Nog, the land of the ever-young of Celtic mythology), which is a homeobox transcription factor required for the maintenance of the undifferentiated state of pluripotent stem cells.  Nanog expression counteracts the differentiation-promoting signals induced by the extrinsic factors LIF (Leukemia Inhibitory Factor) and BMP (Bone Morphogenic Protein).  When Nanog expression is down-regulated, cell differentiation can proceed.  Proteins that regulate Nanog expression include transcription factors Oct4, SOX2, FoxD3, and Tcf3 and tumor suppressor p53.

560109 Rev. 2
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
560109 Rev.2
Citations & References
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Development References (7)

  1. Chambers I, Colby D, Robertson M, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell. 2003; 113:643-655. (Biology). View Reference
  2. Chambers I. The molecular basis of pluripotency in mouse embryonic stem cells. Cloning Stem Cells. 2004; 6(4):386-391. (Biology). View Reference
  3. Ezeh UI, Turek PJ, Reijo RA, Clark AT. Human embryonic stem cell genes OCT4, NANOG, STELLAR, and GDF3 are expressed in both seminoma and breast carcinoma. Cancer. 2005; 104(10):2255-2265. (Biology). View Reference
  4. Mitsui K, Tokuzawa Y, Itoh H, et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell. 2003; 113:631-642. (Biology). View Reference
  5. Pan G, Thomson JA. Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell Res. 2007; 17:42-49. (Biology). View Reference
  6. Sun Y, Li H, Yang H, Rao MS, Zhan M. Mechanisms controlling embryonic stem cell self-renewal and differentiation. Crit Rev Eukaryot Gene Expr.. 2006; 16(3):211-231. (Biology). View Reference
  7. Suzuki A, Raya A, Kawakami Y, et al. Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiation of embryonic stem cells. Proc Natl Acad Sci U S A. 2006; 103(27):10294-10299. (Biology). View Reference
View All (7) View Less
560109 Rev. 2

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.