Overview

  • Product name
    Anti-Nanog antibody - ChIP Grade
    See all Nanog primary antibodies
  • Description
    Rabbit polyclonal to Nanog - ChIP Grade
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-P, ICC, Flow Cyt, ICC/IF, WB, Sandwich ELISA, ChIPmore details
  • Species reactivity
    Reacts with: Human
    Does not react with: Mouse
  • Immunogen

    Synthetic peptide corresponding to Human Nanog aa 29-49.
    Sequence:

    CGPEENYPSLQMSSAEMPHTE

Properties

Applications

Our Abpromise guarantee covers the use of ab21624 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
IHC-P Use at an assay dependent concentration.
ICC 1/100.
Flow Cyt Use at an assay dependent concentration.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF 1/1000.
WB 1/200. Detects a band of approximately 38 kDa (predicted molecular weight: 34 kDa).

Some users report a stronger signal after blocking and diluting antibodies with 1% BSA instead of 5% milk.

Sandwich ELISA Use a concentration of 0.5 µg/ml. For sandwich ELISA, use this antibody as Detection at 0.5 µg/ml with Mouse monoclonal [1E6C4] to Nanog (ab76586) as Capture.
ChIP Use at an assay dependent concentration. PubMed: 18710938

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

  • ab21624 at a 1/1000 dilution staining Nanog in human embryonic stem cells by ICC. An antibody towards Oct4, which has an identical staining pattern to ab21624, was used as a positive control.
  • All lanes : Anti-Nanog antibody - ChIP Grade (ab21624) at 1/200 dilution

    Lane 1 : Human fetal fibroblast lysate
    Lane 2 : Human iPS cell lysate

    Predicted band size: 34 kDa

  • All lanes : Anti-Nanog antibody - ChIP Grade (ab21624) at 1/1000 dilution

    Lane 1 : whole cell extract of mouse 3T3 cells expressing mouse Nanog.
    Lane 2 : whole cell extract of mouse 3T3 cells expressing human Nanog.

    Predicted band size: 34 kDa
    Observed band size: 38 kDa
    why is the actual band size different from the predicted?



    ab21624 recognizes human Nanog but does not recognize mouse Nanog in Western Blot.
  • Immunocytochemical analysis of Human iPSCs labeling Nanog with ab21624 at 1/75 dilution.

    See Abreview

  • ab21624 staining Nanog in Human testis tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 10% serum for 1 hour at 20°C; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody (1/500 in PBS) for 12 hours at 4°C. A Biotin-conjugated Goat anti-rabbit polyclonal (1/200) was used as the secondary antibody.

    See Abreview

  • Standard Curve for Nanog (Analyte: Nanog protein (ab50053)); dilution range 1 pg/ml to 1 ug/ml using Capture Antibody Mouse monoclonal [1E6C4] to Nanog (ab76586) at 0.2 ug/ml and Detector Antibody Rabbit polyclonal to Nanog - ChIP Grade (ab21624) at 0.5 ug/ml.

References

This product has been referenced in:
  • Khabarova AA  et al. Induced pluripotent stem cell line, ICAGi001-A, derived from human skin fibroblasts of a patient with 2p25.3 deletion and 2p25.3-p23.3 inverted duplication. Stem Cell Res 34:101377 (2019). Read more (PubMed: 30616144) »
  • Tong J  et al. Generation of four iPSC lines from peripheral blood mononuclear cells (PBMCs) of an attention deficit hyperactivity disorder (ADHD) individual and a healthy sibling in an Australia-Caucasian family. Stem Cell Res 34:101353 (2019). Read more (PubMed: 30622032) »
See all 144 Publications for this product

Customer reviews and Q&As

11-20 of 21 Abreviews or Q&A

Answer

Gracias por tu respuesta.

Lamento mucho que no hayáis conseguido mejorar los resultados obtenidos. Dado que el anticuerpo está en garantía, si lo deseáis puedo enviaros un reemplazo del mismo anticuerpo (aunque el único lote en stock es el mismo que os hemos enviado esta última vez) o un nuevo anticuerpo contra la misma proteína. Hay numerosas opciones, te mando el link al resultado de la búsqueda para que las consideréis.

https://www.abcam.com/index.html?pageconfig=searchresults&search=Nanog&pt=1&sk=app&sv=69&sn=WB&l=2&fViewMore=1

Si lo preferís puedo enviaros en su lugar un reembolso o vale sin fecha de caducidad para futuras compras en Abcam.

Quedo a la espera de tu respuesta para que me indiques cómo proceder.

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Answer

Muchas gracias por enviarnos el cuestionario relleno y las imágenes de los blots. Lamento muchísimo que el anticuerpo no os haya dado buenos resultados, y después de haber estudiado el protocolo me gustaría haceros algunas sugerencias y preguntas para intentar mejorarlos.

No acabo de entender muy bien la imagen enviada, ¿sería posible ver una imagen completa de la membrana, con los marcadores de peso molecular, y la información acerca del contenido de cada calle? ¿A que corresponden los distintos experimentos?

-Dado que la proteína se expresa a nivel nuclear, un lisado de la fracción nuclear daría probablemente resultados más limpios e intensos correspondientes a la proteína de interés.

-Reducir la carga de proteína suele ser eficaz para reducir también el fondo.

-Para este anticuerpo hemos comprobado que 1%BSA como agente de bloqueo genera una señal más limpia que la leche, y la dilución óptima de trabajo del mismo está entre 1/300-1/500.

En cualquier caso, al estar al anticuerpo bajo garantía (por haberse comprado en los últimos 6 meses), en caso de que las sugerencias no consiguieran optimizar el protocolo, estaré encantada de ofreceros un reemplazo del mismo, o un vale sin caducidad si preferís.

Quedo a la espera de su respuesta. Que pase una buena tarde.

Read More

Question
Answer

Thank you for contacting us. I am sorry that this antibody has been giving no signal in WB. I would like to ask you a few questions about your protocol and offer a few suggestions for improving your results:
1) What type of mouse cells were you testing? What was the cell line or tissue type? Were the samples reduced and boiled prior to loading?
2) Could you please further explain what the different lanes in your blot represent? Was this an IP experiment? If so, could you please specify which antibodies were used for pull down, and which were used for blotting?
3) For western blotting, we have found that using 1% BSA as a blocking agent gives a stronger signal that milk. This antibody may also work better at a higher concentration (1:300 - 1:500) with an overnight incubation at 4C.
I hope this helps, if not, please let me know and I will be happy to assist you further.

Read More
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (human osteosarcoma cell line SAOS2)
Specification
human osteosarcoma cell line SAOS2
Fixative
Paraformaldehyde
Permeabilization
Yes - PBS TRITON .25%
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Apr 20 2012

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cultured Cells (human ES cell line HUES-7 grown on MEFs)
Specification
human ES cell line HUES-7 grown on MEFs
Fixative
Paraformaldehyde
Permeabilization
Yes - see blocking buffer
Blocking step
1% serum,0.1% triton-x,0.1% BSA in PBS as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: rt°C

Abcam user community

Verified customer

Submitted Jan 17 2011

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Testis)
Specification
Testis
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric buffer, pH6
Permeabilization
No
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Sep 13 2010

Application
Flow Cytometry
Sample
Human Cell (Human embryonic carcinoma)
Specification
Human embryonic carcinoma
Preparation
Cell harvesting/tissue preparation method: Trypsin/EDTA
Sample buffer: PBS
Fixation
Paraformaldehyde
Permeabilization
Yes - Saponin
Gating Strategy
Cells were identified by light scatter (forward verse side)

Dr. Mark Jones

Verified customer

Submitted Dec 13 2008

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (H1 ES cell line and NP1 neural progenitors derived)
Specification
H1 ES cell line and NP1 neural progenitors derived
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.4% Triton X-100 in PBS, 15 min
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Sep 19 2008

Application
Western blot
Sample
Human Cell lysate - whole cell (Human embryonic stem cells)
Loading amount
60 µg
Specification
Human embryonic stem cells
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 1%

Abcam user community

Verified customer

Submitted Oct 20 2006

Question
Answer

Thank you for your enquiry. Free or trial sized samples are considered on a case by case basis. On this occasion unfortunately I cannot offer you a free sample. Our policy at Abcam is that if an antibody does not work as specified on the datasheet, we will offer a replacement or reimbursement. Should you decide to go ahead and purchase this product, please let us know how you get on by submitting an Abreview and in return we will offer you 50 Abcam Points which can be redeemed on a number of rewards (a further 100 points will be offered for an image). Please do not hesitate to contact me should ou require further assistance.

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11-20 of 21 Abreviews or Q&A

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