Recombinant
RabMAb

Recombinant Anti-Nanog antibody [EPR20694] - ChIP Grade (ab214549)

Overview

  • Product name

    Anti-Nanog antibody [EPR20694] - ChIP Grade
    See all Nanog primary antibodies
  • Description

    Rabbit monoclonal [EPR20694] to Nanog - ChIP Grade
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ChIP, Flow Cyt, IHC-P, WB, ICC/IF, IPmore details
  • Species reactivity

    Reacts with: Mouse
  • Immunogen

    Recombinant full length protein within Mouse Nanog aa 1 to the C-terminus. The exact sequence is proprietary.
    Database link: Q80Z64

  • Positive control

    • WB: F9 and ES-D3 whole cell lysates. IHC-P: Mouse E14.5 testis tissue. ICC/IF: F9 cells. Flow Cyt: F9 cells. IP: F9 whole cell lysate. ChIP: Chromatin prepared from F9 cells.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab214549 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ChIP Use 5 µg for 25 µg of chromatin.
Flow Cyt 1/60.
IHC-P 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB 1/1000. Detects a band of approximately 29-42 kDa (predicted molecular weight: 34 kDa).
ICC/IF 1/100.
IP 1/30.

Target

  • Function

    Transcription regulator involved in inner cell mass and embryonic stem (ES) cells proliferation and self-renewal. Imposes pluripotency on ES cells and prevents their differentiation towards extraembryonic endoderm and trophectoderm lineages. Blocks bone morphogenetic protein-induced mesoderm differentiation of ES cells by physically interacting with SMAD1 and interfering with the recruitment of coactivators to the active SMAD transcriptional complexes (By similarity). Acts as a transcriptional activator or repressor (By similarity). Binds optimally to the DNA consensus sequence 5'-TAAT[GT][GT]-3' or 5'-[CG][GA][CG]C[GC]ATTAN[GC]-3' (By similarity). When overexpressed, promotes cells to enter into S phase and proliferation.
  • Tissue specificity

    Expressed in testicular carcinoma and derived germ cell tumors (at protein level). Expressed in fetal gonads, ovary and testis. Also expressed in ovary teratocarcinoma cell line and testicular embryonic carcinoma. Not expressed in many somatic organs and oocytes.
  • Sequence similarities

    Belongs to the Nanog homeobox family.
    Contains 1 homeobox DNA-binding domain.
  • Developmental stage

    Expressed in embryonic stem (ES) and carcinoma (EC) cells. Expressed in inner cell mass (ICM) of the blastocyst and gonocytes between 14 and 19 weeks of gestation (at protein level). Not expressed in oocytes, unfertilized oocytes, 2-16 cell embryos and early morula (at protein level). Expressed in embryonic stem cells (ES). Expression decreases with ES differentiation.
  • Cellular localization

    Nucleus.
  • Information by UniProt
  • Database links

  • Alternative names

    • Embryonic stem cell specific homeobox protein (Nanog) antibody
    • ENK antibody
    • FLJ12581 antibody
    • hNanog antibody
    • Homeobox protein NANOG antibody
    • Homeobox transcription factor Nanog antibody
    • homeobox transcription factor Nanog-delta 48 antibody
    • NANOG antibody
    • Nanog homeobox antibody
    • NANOG_HUMAN antibody
    see all

Images

  • Chromatin was prepared from F9 cells according to the Abcam X-ChIP protocol. Cells were fixed with 1% formaldehyde for 10 minutes. The ChIP was performed with 25 µg of chromatin, 5µg of ab214549 (red), and 20 µl of protein A/G sepharose beads slurry (10 µl of sepharose A beads + 10 µl of sepharose G beads). Then 5 μg of rabbit normal IgG was added to the control beads (grey). The immunoprecipitated DNA was quantified by real time PCR (SYBR green chemistry).

  • All lanes : Anti-Nanog antibody [EPR20694] - ChIP Grade (ab214549) at 1/1000 dilution

    Lane 1 : F9 (Mouse embryonic testicular cancer cell line) whole cell lysate
    Lane 2 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Developed using the ECL technique.

    Predicted band size: 34 kDa
    Observed band size: 29-42 kDa
    why is the actual band size different from the predicted?


    Exposure time: 70 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

    The multiple bands observed are consistent with the literature (PMID: 24936455). Negative control: NIH/3T3 (PMID: 12787505).

  • Anti-Nanog antibody [EPR20694] - ChIP Grade (ab214549) at 1/1000 dilution + ES-D3 (mouse embryonic multipotent stem cell line) whole cell lysate at 10 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Developed using the ECL technique.

    Predicted band size: 34 kDa
    Observed band size: 29-42 kDa why is the actual band size different from the predicted?


    Exposure time: 3 minutes


    Blocking/Dilution buffer: 5% NFDM/TBST.

    The multiple bands observed are consistent with the literature (PMID: 24936455).

  • Immunohistochemical analysis of paraffin-embedded mouse E14.5 testis tissue labeling Nanog with ab214549 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Mainly nuclear staining is observed in testis of mouse embryo E14.5 (PMID: 15939376; PMID: 12787505). Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

    Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded adult mouse testis tissue labeling Nanog with ab214549 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Negative tissue: No staining on adult mouse testis is observed(PMID: 12787505; PMID: 15939376).

    Counter stained with Hematoxylin.

    Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

     

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeablized F9 (mouse embryonic testicular cancer cell line) and NIH/3T3 (mouse embryo fibroblast cell line) cells labeling Nanog with ab214549 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in F9 cell line. Negative control: NIH/3T3 (PMID: 17352742).

    The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) (red) at 1/200 dilution.

    Control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

  • Flow cytometric analysis of 4 % paraformaldehyde-fixed, 90% methanol permeabilized F9 (mouse embryonic testicular cancer cell line, Right) and NIH/3T3 (mouse embryo fibroblast cell line, Left) cells labeling Nanog with ab214549 at 1/60 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

    Negative control: NIH/3T3 cell line.

     

  • Nanog was immunoprecipitated from 0.35 mg of F9 (mouse embryonic testicular cancer cell line) whole cell lysate with ab214549 at 1/1000 dilution. Western blot was performed from the immunoprecipitate using ab214549 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5,000 dilution

    Lane 1: F9 whole cell lysate 10 μg (input)

    Lane 2: ab214549 IP in F9 whole cell lysate.

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab214549 in F9 whole cell lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    Exposure time: 15 seconds.

    The multiple bands observed are consistent with the literature (PMID: 24936455).

References

ab214549 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cultured Cells (mouse embryonic stem cells)
Specification
mouse embryonic stem cells
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 23°C
Fixative
Methanol

Malgorzata Borkowska

Verified customer

Submitted Apr 14 2019

Application
IHC - Wholemount
Sample
Mouse Tissue (whole mount E9.5 mouse embryo)
Specification
whole mount E9.5 mouse embryo

Malgorzata Borkowska

Verified customer

Submitted Apr 04 2019

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