Nanog Homeobox (NANOG) antibody

Details for Product No. ABIN967670
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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. ABIN967670
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Immunogen Human Nanog Recombinant Protein
Clone N31-355
Isotype IgG1, kappa
Characteristics 1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. Please refer to us for technical protocols.
3. Triton is a trademark of the Dow Chemical Company.
4. Alexa Fluor is a registered trademark of Molecular Probes, Inc., Eugene, OR.
5. 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.
Purification Purified from tissue culture supernatant or ascites by affinity chromatography.
Alternative Name Nanog
Background The N31-355 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. Nanog is one of the factors that can contribute to reprogramming of differentiated cells to an induced pluripotent stem cell state.
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, and wash (one to two times) with 100 myl of 1× PBS.
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).
4. Remove the fixative from the wells, and wash the wells (one to two times) with 100 myl of 1× PBS.
5. Permeabilize the cells using either cold methanol (a), Triton™ X-100 (b), or Saponin (c): a. Add 100 µl of -20°C 90% methanol to each well and incubate for 5 minutes at RT. b. Add 100 µl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT. c. Add 100 µl of 1× Perm/Wash buffer to each well and incubate for 15 to 30 minutes at RT. Continue to use 1× Perm/Wash buffer for all subsequent wash and dilutions steps.
6. Remove the permeabilization buffer from the wells, and wash one to two times with 100 myl of appropriate buffer (either 1× PBS or 1× Perm/Wash buffer, see step 5.c.).
7. Optional blocking step: Remove the wash buffers, and block the cells by adding 100 µl of blocking buffer or 3% FBS in appropriate dilution buffer to each well and incubating for 15 to 30 minutes at RT.
8. Dilute the antibody to its optimal working concentration in appropriate dilution buffer. Titrate purified (unconjugated) antibodies and second-step reagents to determine the optimal concentration. If using a Bioimaging Certified antibody conjugate, dilute it 1:10.
9. Add 50 µl of diluted antibody per well and incubate for 60 minutes at RT. Incubate in the dark if using fluorescently labeled antibodies.
10. Remove the antibody, and wash the wells three times with 100 myl of wash buffer. An optional detergent wash (100 myl of 0.05% Tween in 1× PBS) can be included prior to the regular wash steps.
11. If the antibody being used is fluorescently labeled, then move to step 12. Otherwise, if using a purified unlabeled antibody, repeat steps 8 to 10 with a fluorescently labeled second-step reagent to detect the purified antibody.
12. After the final wash, 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.
13. 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 cells. 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) Immunoflourescent staining of Nanog in human embryonic stem cells. H9 human ES cells (WiCell Madison, WI) passage 31 grown in mTESR™1 media (StemCell Technologies) on BD Matrigel™ hESC-qualified Matrix were fixed, permeabilized, and stained with Purified Mouse anti-Human Nanog monoclonal antibody (pseudo colored green) at 1.2 ug/mL. The second step reagent was Alexa Fluor® 488 goat anti-mouse Ig (Life Technologies) and counter-staining was with Hoechst 33342 (pseudo-colored blue). The images were captured on a BD Pathway™ 435 Cell Analyzer and merged using BD Attovision™ Software. Permeabilization using 1x BD Perm/Wash™ Buffer (Cat No. 554723) was used for this antibody, Triton™ X-100 or ice-cold methanol is also suitable for permeabilization.
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).

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).

Yu, Vodyanik, Smuga-Otto et al.: "Induced pluripotent stem cell lines derived from human somatic cells." in: Science (New York, N.Y.), Vol. 318, Issue 5858, pp. 1917-20, 2007 (PubMed).

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