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Nanog Homeobox (NANOG) antibody

Details for Product No. ABIN967661
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Antigen
Synonyms 2410002E02Rik, ENK, ecat4
Reactivity
Human
(160), (42), (20), (15), (3), (1), (1)
Host
Mouse
(122), (51), (12), (3)
Clonality (Clone)
Monoclonal ()
Conjugate
Un-conjugated
(7), (6), (6), (5), (5), (5), (2), (2), (1), (1), (1), (1), (1), (1)
Application
Western Blotting (WB), BioImaging (BI)
(147), (100), (40), (34), (31), (23), (18), (16), (10), (5), (1), (1)
Pubmed 7 references available
Quantity 0.1 mg
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Catalog No. ABIN967661
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Immunogen Human Nanog Recombinant Protein
Clone L96-549
Isotype IgG1, kappa
Characteristics 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 us 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.
Purification Purified from tissue culture supernatant or ascites by affinity chromatography.
Alternative Name Nanog
Background 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.
Molecular Weight 36-37 kDa
Research Area Embryogenesis, Embryonic stem cells, Cell Cycle
Application Notes Bioimaging:
1. Seed the cells in appropriate culture medium at an appropriate cell density in an 96-well Imaging Plate , and culture overnight to 48 hours.
2. Remove the culture medium from the wells, wash the wells twice with 100 myl of 1× PBS, and fix the cells by adding 100 µl of fresh 3.7% Formaldehyde in PBS or fixation buffer 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 myl of 1× PBS.
4. Permeabilize the cells by adding 100 µl of 1× Perm/Wash™ buffer to each well and incubating for 30 minutes at RT.
5. Remove the permeabilizer, and wash the wells twice with 100 myl of 1× PBS.
6. Dilute the antibody in 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 myl of 1× PBS.
8. Remove the PBS, dilute the second-step reagent in 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 myl of 1× PBS.
10. Remove the PBS, and counter-stain the nuclei by adding 100 ml of a 2 mg/ml solution of Hoechst 33342 in 1× PBS to each well at least 15 minutes before imaging.
11. View and analyze the cells on an appropriate imaging instrument.
Comment

Related Products: ABIN967389

Restrictions For Research Use only
Format Liquid
Concentration 0.5 mg/ml
Buffer Aqueous buffered solution.
Preservative Sodium azide
Precaution of Use This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Storage 4 °C
Supplier Images
anti-Nanog Homeobox (NANOG) antibody 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.
anti-Nanog Homeobox (NANOG) antibody (2) 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 (ABIN968902 or ABIN968903) was the gel-loading control (lower blot).
anti-Nanog Homeobox (NANOG) antibody (3) 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.
Product cited in: Mitsui, Tokuzawa, Itoh et al.: "The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells." in: Cell, Vol. 113, Issue 5, pp. 631-42, 2003 (PubMed).

Chambers, Colby, Robertson et al.: "Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells." in: Cell, Vol. 113, Issue 5, pp. 643-55, 2003 (PubMed).

Chambers: "The molecular basis of pluripotency in mouse embryonic stem cells." in: Cloning and stem cells, Vol. 6, Issue 4, pp. 386-91, 2005 (PubMed).

Ezeh, Turek, Reijo et al.: "Human embryonic stem cell genes OCT4, NANOG, STELLAR, and GDF3 are expressed in both seminoma and breast carcinoma." in: Cancer, Vol. 104, Issue 10, pp. 2255-65, 2005 (PubMed).

Suzuki, Raya, Kawakami et al.: "Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiation of embryonic stem cells." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 103, Issue 27, pp. 10294-9, 2006 (PubMed).

Sun, Li, Yang et al.: "Mechanisms controlling embryonic stem cell self-renewal and differentiation." in: Critical reviews in eukaryotic gene expression, Vol. 16, Issue 3, pp. 211-31, 2006 (PubMed).

Pan, Thomson: "Nanog and transcriptional networks in embryonic stem cell pluripotency." in: Cell research, Vol. 17, Issue 1, pp. 42-9, 2007 (PubMed).

Validation Images
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