Nanog antibody

Catalog No. ABIN967670

Product Details anti-Nanog Antibody

Target: Nanog (NANOG) (Nanog Homeobox (NANOG))

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This Nanog antibody is un-conjugated

Application: Western Blotting (WB), BioImaging (BI)

Brand: BD Pharmingen™

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: The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

Immunogen: Human Nanog Recombinant Protein

Clone: N31-355

Isotype: IgG1 kappa

Application Details

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

Handling

Format: Liquid

Concentration: 0.5 mg/mL

Buffer: Aqueous buffered solution containing ≤0.09 % sodium azide.

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

Storage Comment: Store undiluted at 4°C.

References for anti-Nanog antibody (ABIN967670)

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, Antosiewicz-Bourget, Frane, Tian, Nie, Jonsdottir, Ruotti, Stewart, Slukvin, Thomson: "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).

Sun, Li, Yang, Rao, Zhan: "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).

Suzuki, Raya, Kawakami, Morita, Matsui, Nakashima, Gage, Rodríguez-Esteban, Izpisúa Belmonte: "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).

Ezeh, Turek, Reijo, Clark: "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).

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

Mitsui, Tokuzawa, Itoh, Segawa, Murakami, Takahashi, Maruyama, Maeda, Yamanaka: "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).

Target Details for Nanog

Target: Nanog (NANOG) (Nanog Homeobox (NANOG))

Alternative Name: Nanog (NANOG Products)

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

Pathways: Stem Cell Maintenance