Oct-3/4 Antibody [Unconjugated]

BG01V human embryonic stem cells were fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. Oct‑3/4/DNA complexes were immunoprecipitated using 5 μg Goat Anti-Human/Mouse Oct‑3/4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1759) or control antibody (Catalog # AB-108-C) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Goat IgG Secondary Antibody (Catalog # BAF109). Immunocomplexes were captured using 50 μL of MagCellect Streptavidin Ferrofluid (Catalog # MAG999) and DNA was purified using chelating resin solution. The nanog promoter was detected by standard PCR.

Alkaline phosphatase (ALPL) and Oct‑3/4 were detected in human BG01V embryonic stem cells using 10 µg/mL Human/Mouse/Rat ALPL Monoclonal Antibody (Catalog # MAB1448) and 10 µg/mL Goat Anti-Human/Mouse Oct‑3/4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1759). Cells were incubated with primary antibodies for 3 hours at room temperature. Cells were stained for ALPL using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (pseudo-stained green; Catalog # NL007), and stained for Oct-3/4 using the NorthernLights 637-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL002). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Simple Western lane view shows lysates of BG01V human embryonic stem cells and NTera‑2 human testicular embryonic carcinoma cell line, loaded at 0.2 mg/mL. A specific band was detected for Oct‑3/4 at approximately 50 kDa (as indicated) using 20 µg/mL of Goat Anti-Human/Mouse Oct‑3/4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1759) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Western blot shows lysates of BG01V human embryonic stem cells and D3 mouse embryonic stem cell line. PVDF membrane was probed with 0.5 µg/mL of Goat Anti-Human/Mouse Oct‑3/4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1759) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for Oct‑3/4 at approximately 48 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

• Reactivity: Hu, Mu
• Applications: WB, Simple Western, IP, ChIP, ICC/IF
• Clonality: Polyclonal
• Host: Goat
• Conjugate: Unconjugated
• Concentration: LYOPH

Oct-3/4 Antibody [Unconjugated] Summary

• Immunogen: E. coli-derived recombinant human Oct‑3/4
  Met1-Asn265 (Met262Leu)
  Accession # Q01860
• Specificity: Detects human Oct-3/4 in direct ELISAs and Western blots.
• Source: N/A
• Isotype: IgG
• Clonality: Polyclonal
• Host: Goat
• Gene: POU5F1
• Purity Statement: Antigen Affinity-purified

Applications/Dilutions

• Dilutions:
  • Western Blot 0.5 ug/mL
  • Simple Western 20 ug/mL
  • Immunoprecipitation 3 ug/10^6 cells
  • Chromatin Immunoprecipitation (ChIP) 5 ug/10^6 cells
  • Immunocytochemistry 5-15 ug/mL

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.2 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Oct-3/4 Antibody [Unconjugated]

• 3-Oct
• Oct-3/4
• Oct4
• Otf3g
• Otf4
• Pou5f1

Background

Oct-3/4, a member of POU transcription factors, was identified as a DNA-binding protein that activates gene transcription via a cis-element containing an octamer motif (1). It is expressed in totipotent embryonic stem and germ cells (2, 3). A critical amount of Oct-3/4 expressed is required to sustain stem cell self-renewal and pluripotency (4). When embryonic stem cells are induced to differentiate, Oct-3/4 is downregulated and this downregulation of Oct-3/4 has proven to be essential for proper and divergent developmental program (5). The Oct-3/4 molecule is not only a master regulator of pluripotency that controls the lineage commitment but also is the most recognized marker used for the identification of totipotent embryonic stem cells.

1. Scholer, H.R. et al. (1990) Nature 344:435.
2. Scholer, H.R. et al. (1989) EMBO J. 8:2543.
3. Rosner, M.H. et al. (1990) Nature 345:686.
4. Niwa, H. et al. (2000) Nat. Genet. 24:372.
5. Pesce, M. et al. (2001) Stem Cells 19:271.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for Oct-3/4 Antibody (AF1759)(25)

Publications using AF1759

• DZ Hazelbaker, A Beccard, G Angelini, P Mazzucato, A Messana, D Lam, K Eggan, LE Barrett A multiplexed gRNA piggyBac transposon system facilitates efficient induction of CRISPRi and CRISPRa in human pluripotent stem cells Sci Rep, 2020;10(1):635. 2020 [PMID: 31959800] (ICC, Human)
• T Hyvärinen, A Hyysalo, FE Kapucu, L Aarnos, A Vinogradov, SJ Eglen, L Ylä-Outine, S Narkilahti Functional characterization of human pluripotent stem cell-derived cortical networks differentiated on laminin-521 substrate: comparison to rat cortical cultures Sci Rep, 2019;9(1):17125. 2019 [PMID: 31748598] (ICC, Rat)
• M Sherman-Sa, H Onallah, A Holth, R Reich, B Davidson SOX2 and SOX9 are markers of clinically aggressive disease in metastatic high-grade serous carcinoma Gynecol. Oncol., 2019;0(0):. 2019 [PMID: 30904337] (ICC, Human)
• B Cao, M Duan, Y Xing, C Liu, F Yang, Y Li, T Yang, Y Wei, Q Gao, J Jiang O-GlcNAc transferase activates stem-like cell potential in hepatocarcinoma through O-GlcNAcylation of eukaryotic initiation factor 4E J. Cell. Mol. Med., 2019;0(0):. 2019 [PMID: 30677218] (Western Blot, Human)
• W Zhang, C Chronis, X Chen, H Zhang, R Spalinskas, M Pardo, L Chen, G Wu, Z Zhu, Y Yu, L Yu, J Choudhary, J Nichols, MM Parast, B Greber, P Sahlén, K Plath The BAF and PRC2 Complex Subunits Dpf2 and Eed Antagonistically Converge on Tbx3 to Control ESC Differentiation Cell Stem Cell, 2019;24(1):138-152.e8. 2019 [PMID: 30609396] (Western Blot, Mouse)
• S Kaur, AG Abu-Shahba, RO Paananen, H Hongisto, H Hiidenmaa, H Skottman, R Seppänen-K, B Mannerströ Small non-coding RNA landscape of extracellular vesicles from human stem cells Sci Rep, 2018;8(1):15503. 2018 [PMID: 30341351] (Differentiation, Human)
• A Buskin, L Zhu, V Chichagova, B Basu, S Mozaffari-, D Dolan, A Droop, J Collin, R Bronstein, S Mehrotra, M Farkas, G Hilgen, K White, KT Pan, A Treumann, D Hallam, K Bialas, G Chung, C Mellough, Y Ding, N Krasnogor, S Przyborski, S Zwolinski, J Al-Aama, S Alharthi, Y Xu, G Wheway, K Szymanska, M McKibbin, CF Inglehearn, DJ Elliott, S Lindsay, RR Ali, DH Steel, L Armstrong, E Sernagor, H Urlaub, E Pierce, R Lührmann, SN Grellschei, CA Johnson, M Lako Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa Nat Commun, 2018;9(1):4234. 2018 [PMID: 30315276] (ICC, Human)
• X Gao, JJ Yourick, RL Sprando Generation of nine induced pluripotent stem cell lines as an ethnic diversity panel Stem Cell Res, 2018;31(0):193-196. 2018 [PMID: 30099336] (ICC, Human)
• K Alsayegh, K Matsuura, H Sekine, T Shimizu Dinaciclib potently suppresses MCL-1 and selectively induces the cell death in human iPS cells without affecting the viability of cardiac tissue Sci Rep, 2017;7(0):45577. 2017 [PMID: 28361959] (ICC, Human)
• Brain tissue banking for stem cells for our future Sci Rep, 2016;6(0):39394. 2016 [PMID: 27991551] (IHC, Human)
• Generation of a TLE3 heterozygous knockout human embryonic stem cell line using CRISPR-Cas9 Stem Cell Res, 2016;17(2):441-443. 2016 [PMID: 27879221] (IHC-Fr, Human)
• Generation of a TLE1 homozygous knockout human embryonic stem cell line using CRISPR-Cas9 Stem Cell Res, 2016;17(2):430-432. 2016 [PMID: 27879218] (IHC-Fr, Human)
• Luo, Lan, Kawakatsu, Miho, Guo, Chao-Wan, Urata, Yoshishi, Huang, Wen-Jing, Ali, Haytham, Doi, Hanako, Kitajima, Yuriko, Tanaka, Takayuki, Goto, Shinji, Ono, Yusuke, Xin, Hong-Bo, Hamano, Kimikazu, Li, Tao-Shen Effects of antioxidants on the quality and genomic stability of induced pluripotent stem cells. Sci Rep, 2014;4(0):3779. 2014 [PMID: 24445363] (Western Blot, Human)
• Jumabay M, Abdmaulen R, Ly A, Cubberly M, Shahmirian L, Heydarkhan-Hagvall S, Dumesic D, Yao Y, Bostrom K Pluripotent stem cells derived from mouse and human white mature adipocytes. Stem Cells Transl Med, 2014;3(2):161-71. 2014 [PMID: 24396033] (ICC, Mouse)
• Wada, Naohisa, Maeda, Hidefumi, Hasegawa, Daigaku, Gronthos, Stan, Bartold, Peter Ma, Menicanin, Danijela, Fujii, Shinsuke, Yoshida, Shinichi, Tomokiyo, Atsushi, Monnouchi, Satoshi, Akamine, Akifumi Semaphorin 3A induces mesenchymal-stem-like properties in human periodontal ligament cells. Stem Cells Dev, 2014;23(18):2225-36. 2014 [PMID: 24380401] (ICC, Human)
• Apostolou E, Ferrari F, Walsh R, Bar-Nur O, Stadtfeld M, Cheloufi S, Stuart H, Polo J, Ohsumi T, Borowsky M, Kharchenko P, Park P, Hochedlinger K Genome-wide chromatin interactions of the Nanog locus in pluripotency, differentiation, and reprogramming. Cell Stem Cell, 2013;12(6):699-712. 2013 [PMID: 23665121] (ChIP, Mouse)
• Hayashi, Ryuhei, Ishikawa, Yuki, Ito, Miyuki, Kageyama, Tomofumi, Takashiba, Kuniko, Fujioka, Tsuyoshi, Tsujikawa, Motokazu, Miyoshi, Hiroyuki, Yamato, Masayuki, Nakamura, Yukio, Nishida, Kohji Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium. PLoS ONE, 2012;7(9):e45435. 2012 [PMID: 23029008] (ICC, Human)
• Wong RC, Pollan S, Fong H, Ibrahim A, Smith EL, Ho M, Laslett AL, Donovan PJ A novel role for an RNA polymerase III subunit POLR3G in regulating pluripotency in human embryonic stem cells. Stem Cells, 2011;29(10):1517-27. 2011 [PMID: 21898682] (ChIP, Human)
• Kubota H, Wu X, Goodyear SM, Avarbock MR, Brinster RL Glial cell line-derived neurotrophic factor and endothelial cells promote self-renewal of rabbit germ cells with spermatogonial stem cell properties. FASEB J., 2011;25(0):2604-14. 2011 [PMID: 21525489] (ICC, Rabbit)
• Chng Z, Teo A, Pedersen RA, Vallier L SIP1 mediates cell-fate decisions between neuroectoderm and mesendoderm in human pluripotent stem cells. Cell Stem Cell, 2010;6(1):59-70. 2010 [PMID: 20074535] (ChIP, Human)
• Fong H, Hohenstein KA, Donovan PJ Regulation of self-renewal and pluripotency by Sox2 in human embryonic stem cells. Stem Cells, 2008;26(8):1931-8. 2008 [PMID: 18388306] (ICC, Human)
• Ware CB, Nelson AM, Blau CA Controlled-rate freezing of human ES cells. BioTechniques, 2005;38(6):879-80, 882-3. 2005 [PMID: 16018548] (ICC, Human)
• Rajesh</LastName><ForeNam D</Initial, Rajesh D, Chinnasamy N, Mitalipov SM, Wolf DP, Slukvin I, Thomson JA, Shaaban AF Differential requirements for hematopoietic commitment between human and rhesus embryonic stem cells. Stem Cells, 2007;25(2):490-9. 2007 [PMID: 17284653] (Flow Cytometry, Human)
• Ware CB, Nelson AM, Blau CA A comparison of NIH-approved human ESC lines. Stem Cells, 2006;24(12):2677-84. 2006 [PMID: 16916927] (ICC, Human)

Bioinformatics

• Gene Symbol: POU5F1
• Uniprot:
  • Q01860()