Overview

  • Product name
  • Description
    Rabbit polyclonal to Oct4
  • Host species
    Rabbit
  • Specificity
    The antibody reacts with 45 kDa Oct4 (also designated Oct3A) and in a less extent 33 kDa Oct3B from human, mouse, and rat samples.
  • Tested applications
    Suitable for: IHC-Fr, WB, IP, ICC/IF, Flow Cyt, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Pig, Macaque monkey
    Predicted to work with: Chimpanzee
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive).
    (Peptide available as ab52110)

  • Positive control
    • Rat kidney tissue lysate can be used as a positive control.

Properties

Applications

Our Abpromise guarantee covers the use of ab18976 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-Fr Use a concentration of 20 µg/ml.
WB Use a concentration of 1 - 2 µg/ml. Predicted molecular weight: 45 kDa.Can be blocked with Oct4 peptide (ab52110).
IP Use a concentration of 20 µg/ml.
ICC/IF Use at an assay dependent concentration. PubMed: 19736565
Flow Cyt Use at an assay dependent concentration. PubMed: 17719011

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

IHC-P 1/100. Perform enzymatic antigen retrieval before commencing with IHC staining protocol.

Target

  • Function
    Transcription factor that binds to the octamer motif (5'-ATTTGCAT-3'). Forms a trimeric complex with SOX2 on DNA and controls the expression of a number of genes involved in embryonic development such as YES1, FGF4, UTF1 and ZFP206. Critical for early embryogenesis and for embryonic stem cell pluripotency.
  • Tissue specificity
    Expressed in developing brain. Highest levels found in specific cell layers of the cortex, the olfactory bulb, the hippocampus and the cerebellum. Low levels of expression in adult tissues.
  • Sequence similarities
    Belongs to the POU transcription factor family. Class-5 subfamily.
    Contains 1 homeobox DNA-binding domain.
    Contains 1 POU-specific domain.
  • Developmental stage
    Highly expressed in undifferentiated embryonic stem cells and expression decreases gradually after embryoid body (EB) formation.
  • Domain
    The POU-specific domain mediates interaction with PKM2.
  • Post-translational
    modifications
    Sumoylation enhances the protein stability, DNA binding and transactivation activity. Sumoylation is required for enhanced YES1 expression.
    Ubiquitinated; undergoes 'Lys-63'-linked polyubiquitination by WWP2 leading to proteasomal degradation.
  • Cellular localization
    Nucleus. Expressed in a diffuse and slightly punctuate pattern.
  • Information by UniProt
  • Database links
  • Alternative names
    • Octamer binding transcription factor 4 antibody
    • MGC22487 antibody
    • Oct 3 antibody
    • Oct 4 antibody
    • Oct-3 antibody
    • Oct-4 antibody
    • OCT3 antibody
    • Oct4 antibody
    • Octamer binding protein 3 antibody
    • Octamer binding protein 4 antibody
    • Octamer binding transcription factor 3 antibody
    • Octamer-binding protein 3 antibody
    • Octamer-binding protein 4 antibody
    • Octamer-binding transcription factor 3 antibody
    • OTF 3 antibody
    • OTF 4 antibody
    • OTF-3 antibody
    • OTF3 antibody
    • OTF4 antibody
    • PO5F1_HUMAN antibody
    • POU class 5 homeobox 1 antibody
    • POU domain class 5 transcription factor 1 antibody
    • POU domain transcription factor OCT4 antibody
    • POU domain, class 5, transcription factor 1 antibody
    • POU-type homeodomain-containing DNA-binding protein antibody
    • POU5F1 antibody
    see all

Images

  • Immunohistochemistry analysis of paraffin-embedded human breast tissues (5 µm thick) labeling Oct4 with ab18976 at 1/100 dilution.

    Tissues were rehydrated in deionized water and incubated in PBS prior to per meabilization in 0.1% PBS-TritonX 100 and overnight incubation with ab18976 in a humid chamber.Washing in PBS and secondary antibody (Alexa Fluor®) incubation for 2h was followed by a final wash and mounting.

    Blue: DAPI stain for nuclei.

    Minimal and mostly cytoplasmic expression was seen in the normal resting breast (left panel). During normal lactation (right panel), OCT4 was upregulated and expressed in both the nucleus and cytoplasm.

  • Anti-Oct4 antibody (ab18976) at 1/500 dilution + mouse embryonic stem cell whole cell lysate at 100 µg/ml

    Secondary
    HRP conjugated goat anti-rabbit

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 45 kDa
    Observed band size: 45 kDa
    Additional bands at: 25 kDa (possible non-specific binding), 60 kDa (possible non-specific binding)


    Exposure time: 1 minute


    The blot was blocked with 5% milk for 1 hour at RT and inbubated with the primary antibody for 16 hours at 4°C.

    See Abreview

  • Immunohistochemical analysis of paraffin embedded Human breast tissues (5 µm thick) labeling Oct4 with ab18976 at 1/100 to 1/200 dilution. Heat induced epitope retrieval was performed at 100°C for 30 min. Slides were counterstained with hematoxylin.

    Minimal and mostly cytoplasmic expression was seen in the normal resting breast (left panel). During normal lactation (right panel), OCT4 was upregulated and expressed in both the nucleus and cytoplasm.

  • ab18976 at 1/100 dilution staining Oct-4 in mouse brain tissue by immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). Sections were paraformaldehyde fixed prior to blocking in 5% BSA for 1 hour at 25°C and then incubated with ab18976 for 12 hours at 4°C. A Cy3 conjugated donkey polyclonal to rabbit Ig, diluted 1/500, was used as the secondary antibody.

    See Abreview

  • Anti-Oct4 antibody (ab18976) at 0.5 µg/ml + Lysates prepared from rat kidney tissue.

    Predicted band size: 45 kDa



    The antibody reacts with 45 kDa Oct4 (also designated Oct-3A) and in a less extent 33 kDa Oct-3B from human, mouse, and rat samples.

References

This product has been referenced in:
  • Baird A  et al. Osteoblast differentiation of equine induced pluripotent stem cells. Biol Open 7:N/A (2018). Read more (PubMed: 29685993) »
  • Li C  et al. The three branches of the unfolded protein response exhibit differential significance in breast cancer growth and stemness. Exp Cell Res 367:170-185 (2018). IHC-P . Read more (PubMed: 29601799) »
See all 83 Publications for this product

Customer reviews and Q&As

1-10 of 21 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry
Sample
Mouse Cultured Cells (Neural stem cells)
Permeabilization
No
Specification
Neural stem cells
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Fixative
Paraformaldehyde

Kyungjoo Seong

Verified customer

Submitted Oct 31 2016

Application
Flow Cytometry
Sample
Human Cell (H9 hESCs)
Permeabilization
Yes - 0.25% Triton X-100 in DPBS
Gating Strategy
Fibroblasts (white)
Specification
H9 hESCs
Preparation
Cell harvesting/tissue preparation method: Accutase
Sample buffer: PBS
Fixation
Paraformaldehyde

Abcam user community

Verified customer

Submitted Sep 24 2015

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Dog Tissue sections (Fetal Brain)
Antigen retrieval step
Other
Permeabilization
No
Specification
Fetal Brain
Blocking step
Dako protein block as blocking agent for 10 minute(s) · Concentration: .25% · Temperature: 20°C
Fixative
Formaldehyde

Ms. Erica Sloma

Verified customer

Submitted Oct 09 2014

Question

Dear Tech Support Team,



Please read below the details of customer's complaint:



Antibody code: ab18976

General Information:

Antibody storage conditions (temperature/reconstitution etc) (-20).

Description of the problem (high background, low signal, non-specific staining etc.) no staining in ImmunoFluorescence staning.

Sample (Species/Tissue/Cell Type/Cell Line etc.) Human & mouse cells & tissue

Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.) Cells: methanol, Human tissue: formaldehyde, Mouse tissue: bouin.

Antigen retrieval (Enzymatic method, Heat mediated technique etc.) Urea

Permeabilization step

Blocking conditions (Buffer/time period, Blocking agent etc.) Fetal Calf Serum in PBSX1 for 40mins

Primary Antibody (Diluent/Dilution/Incubation time, Wash step)1:100 and 1:200 in blocking buffer for over night at 4C and washes with PBSX1 for 3 times, 5mins each.

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Fluorescent Anti Rabbit-1:1200 in blocking buffer for 40mins at room tempreture and washes in PBSX1 for 3 times, 10mins each.

Detection method fluorescent microscope

Positive and negative controls used (please specify) negative control: tissue/cells incubated with the secondary antibody without the first one.

Positive control: tissues/cells stained with another antibody that works excellent (Sall-4 of abcam: ab

Optimization attempts (problem solving):

How many times have you tried the IHC? Our lab: since many years. Me: 2 years

Have you run a "No Primary" control? Yes

Do you obtain the same results every time? Yes

What steps have you altered? All the steps are the same.



The attached images:

http://mail.zotal.co.il/exchange/Alla/Inbox/%D7%A6%D7%91%D7%99%D7%A2%D7%AA%20OCT%20%D7%91%D7%A8%D7%A7%D7%9E%D7%AA%20HUMAN.EML/1_multipart_xF8FF_3_Merged-30%2B31.jpg/C58EA28C-18C0-4a97-9AF2-036E93DDAFB3/Merged-30%2B31.jpg?attach=1 and http://mail.zotal.co.il/exchange/Alla/Inbox/%D7%A6%D7%91%D7%99%D7%A2%D7%AA%20OCT%20%D7%91%D7%A8%D7%A7%D7%9E%D7%AA%20HUMAN.EML/1_multipart_xF8FF_2_Merged-27%2B28.jpg/C58EA28C-18C0-4a97-9AF2-036E93DDAFB3/Merged-27%2B28.jpg?attach=1 - human sections, same dilutions, looks like the negative control



All other attached images - mouse sections, 1/200 and 1/100, looks like the negative control



Please advise.



Thanks in advance.

Have a nice week,

Read More
Answer

Thank you and your customer for taking the time to complete our questionnaire and contact us. I am sorry to hear your customer has had difficulty obtaining satisfactory results from this antibody. The details your customer has kindly provided will enable us to investigate this case for your customer and this is also helpful in our records for monitoring of quality.
Although the other antibody has worked well using this procedure, individual antibodies will often require optimization especially if the target is different. I would like to offer some suggestions to help optimise the results from ab18976.
1.) I can recommend to add a permeabilization step to the protocol. Oct4 is in the nucleus. The following detergents can be used: Triton or NP-40 Use 0.1 to 0.2% in PBS for 10 min only. These will partially dissolve the nuclear membrane and are therefore very suitable for nuclear antigen staining.
2.) Oct4 is expressed in developing brain. Highest levels are found in specific cell layers of the cortex, the olfactory bulb, the hippocampus and the cerebellum. Highly expressed in undifferentiated embryonic stem cells and expression decreases gradually after embryoid body (EB) formation. Since there is only a low level of expression in adult tissues, I suggest to run a positive control.
I would also appreciate if your customer can confirm some details of the procedure.
1.) What tissues and cells were used?
2.) What does it urea mean as antigen retrieval step?
3.) What cells are shown in the images?
We are happy to offer this technical support. In the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.
I hope this information is helpful, thank you and your customer for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Read More

Answer

Thank you for your enquiry and your interest.

I can confirm that two isoforms of Oct 4 have been identified, and the exact amino acid sequences (aa) can be found at this website: http://www.uniprot.org/uniprot/Q01860

- Isoform A: Q01860-1 (1-360 aa)

- Isoform B: Q01860-2 (1-265 aa)

ab109183 - this antibody was raised against a peptide corresponding to the region between 265-360 aa, soit is very likely to recognize Isoform A.

ab18976 - the immunogen selected was around region 1-14 aa, thus it would recognize potentially both Isofm A and B.

If you need any further assistance in the future, please do not hesitate to contact me.

Read More

Answer

Muchas gracias por tu respuesta.

Es importante que todos los anticuerpos que escojas para llevar a cabo IHC-P en tejido humano estén validados (y por tanto garantizados por Abcam) para funcionar en esta especie y aplicación. Esta información aparece en la hoja técnica de los productos.

También es importante echar un vistazo a las hojas de datos de cada uno de los productos para tener en cuenta recomendaciones especificas, tales como diluciones, métodos de recuperación antigénica, y demás que pueden aparecer en ellas. A las fichas técnicas se puede acceder desde la página web, escribiendo el código del anticuerpo (ab955, por ejemplo) en la casilla azul en la parte de arriba de nuestra página web (https://www.abcam.com/).

Desconozco las combinaciones que queréis llevar a cabo mediante inmunofluorescencia doble con los anticuerpos mencionados. En general, a la hora de hacer inmunoensayos múltiples hay que tener mucha precaución para evitar reactividad cruzada. Es importante usar anticuerpos procedentes de distintas especies si se puede, o al menos con diferentes isotipos. Una buena forma de evitar reactividad cruzada es usando secundarios de la misma especie. Además esto permite usar suero de dicha especie para bloquear.

Te copio los links a los protocolos que tenemos en nuestra web para llevar a cabo doble inmunofluorescencia por s pudieran resultar de ayuda:

https://www.abcam.com/index.html?pageconfig=resource&rid=11459

https://www.abcam.com/index.html?pageconfig=resource&rid=11458

De todas maneras si quieres que comentemos distintas posibilidades y escenarios posibles, no dudes en contactarme otra vez.

Respecto a los secundarios, tenemos muchos anticuerpos que podrían serviros. Te aconsejo que en función de los primarios y combinaciones que vayáis a llevar a cabo, optéis por uno u otro. Para llevar a cabo la búsqueda de los secundarios desde la pagina web (https://www.abcam.com/) pincha en la pestaña “Secondary Antibodies”. Se abre una nueva ventana de “Advance Search”, en la cual se pueden elegir las categorías requeridas. La única categoría obligatoria es el isotipo del anticuerpo, pues tiene que coincidir con el del primario. El resto de categorías son opcionales, pero son muy útiles para afinar la búsqueda y elegir el anticuerpo más apropiado según la especie del primario, la conjugación deseada, la clonalidad, la aplicación en la que se va a testar, etc…

Los precios y la disponibilidad pueden consultarse igualmente en la parte superior de la datasheet de los productos. Si necesitas un presupuesto no dudes en hacérmelo saber para que te lo envíe. Además tenemos algún descuento para pedidos superiores a 5 productos, por lo que si vais a realizar un pedido de estas características te animaría a que nos contactaras primero.

Espero que esta información sea de ayuda. Como te digo, para cualquier aclaración o más sugerencias, estaré encantada de echar una mano.

Read More

Answer

Thank you for contacting us.

Please advice customer to consult publication whole selecting the antibodies.

HEK293T cells do not express c-Myc and Oct4. They express SOX2.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1289416/

Human embryonic fibroblasts have basal level of Oct4 and Sox2 proteins. You may need to induce the expression of these proteins.

http://sklrm.shsmu.edu.cn/manage/edit/UploadFile/200982102734474.pdf

c-myc is also OK in HEF cells; http://www.pnas.org/content/106/31/12759.full

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More
Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Cat Tissue sections (epithelial cancer, small intestine, testis)
Specification
epithelial cancer, small intestine, testis
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate
Permeabilization
No
Blocking step
universal blocking reagent as blocking agent for 10 minute(s) · Concentration: 100% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Nov 30 2011

Answer

Thank you very much for your email. I apologize for this confusion. Unfortunately I do not know where it comes from. I can however confirm that the immunogen used for this antibody is from amino acid 1 to 14. The sequence is AGHLASDFAFSPC. I hope this information is helpful. Please do not hesitate to contact us again should you have any other question or concern.

Read More
Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Dog Tissue sections (canine mast cell tumor)
Specification
canine mast cell tumor
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate, high pH, and protease
Permeabilization
No
Blocking step
universal reagent as blocking agent for 10 minute(s) · Concentration: 100% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Oct 24 2011

1-10 of 21 Abreviews or Q&A

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