Recombinant Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR17929] to Oct4 - ChIP Grade
- Suitable for: WB, ICC/IF, IP, IHC-P, ChIP-sequencing, ChIP, Flow Cyt (Intra)
- Reacts with: Mouse, Human
Related conjugates and formulations
Overview
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Product name
Anti-Oct4 antibody [EPR17929] - ChIP Grade
See all Oct4 primary antibodies -
Description
Rabbit monoclonal [EPR17929] to Oct4 - ChIP Grade -
Host species
Rabbit -
Specificity
Oct4 is highly expressed in undifferentiated embryonic stem cells and cancer stem cell-like cells (PMID: 26013162, 21826175). ab181557 can’t detect the target band in undifferentiated cancer cell lines with low expression level of Oct4, such as HeLa, HEK-293, MDA-MB-231, HepG2, Huh7, HCT-116 and PANC-1 (PMID: 21975933, 29789579, 25625591, 26059097, 23928699, 27344963, 25837691, 29254202, 28854261, 27996162), even at the dilution of 1:200.
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Tested applications
Suitable for: WB, ICC/IF, IP, IHC-P, ChIP-sequencing, ChIP, Flow Cyt (Intra)more details -
Species reactivity
Reacts with: Mouse, Human
Predicted to work with: Rat, Sheep, Horse, Cow, Pig, Common marmoset -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: NCCIT, F9, and NTERA-2 cl.D1 whole cell lysates. IHC-P: Human seminoma and dysgerminoma of ovary tissues. ICC/IF: NCCIT cells. IP: NCCIT whole cell extract. ChIP: Chromatin prepared from F9 cells. ChIP-seq: NCCIT cells. Flow cyto(intra): NCCIT cells.
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General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR17929 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Assay kits
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ChIP Related Products
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Compatible Secondaries
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Conjugation kits
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Isotype control
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab181557 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 |
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WB | (3) |
1/1000. Detects a band of approximately 45 kDa (predicted molecular weight: 39 kDa).
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ICC/IF | (8) |
1/250.
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IP |
1/50.
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IHC-P | (1) |
1/1000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
The mouse recommendation is based on the WB results. We do not guarantee IHC-P for mouse as there is no suitable positive tissue in-house. |
ChIP-sequencing |
Use at an assay dependent concentration.
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ChIP | (1) |
Use 5 µg for 25 µg of chromatin.
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Flow Cyt (Intra) |
Use at an assay dependent concentration.
Purified format. |
Notes |
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WB
1/1000. Detects a band of approximately 45 kDa (predicted molecular weight: 39 kDa). |
ICC/IF
1/250. |
IP
1/50. |
IHC-P
1/1000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. The mouse recommendation is based on the WB results. We do not guarantee IHC-P for mouse as there is no suitable positive tissue in-house. |
ChIP-sequencing
Use at an assay dependent concentration. |
ChIP
Use 5 µg for 25 µg of chromatin. |
Flow Cyt (Intra)
Use at an assay dependent concentration. Purified format. |
Target
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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
modificationsSumoylation 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
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Database links
- Entrez Gene: 282316 Cow
- Entrez Gene: 5460 Human
- Entrez Gene: 18999 Mouse
- Entrez Gene: 100127461 Pig
- Entrez Gene: 294562 Rat
- Omim: 164177 Human
- SwissProt: O97552 Cow
- SwissProt: Q01860 Human
see all -
Alternative names
- Octamer binding transcription factor 4 antibody
- MGC22487 antibody
- Oct 3 antibody
see all
Images
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Flow cytometry overlay histogram showing left NCCIT positive cells and right negative HeLa stained with ab181557 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab181557) (1x 106 in 100μl at 0.2μg/ml (1/10300)) for 30min at 22°C.
The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C
Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.
Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.
This antibody gave a positive signal in NCCIT Fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.
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Chromatin was prepared from F9 (Mouse embyro testicular cancer cell line) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab181557 (red, and 20µl of Anti rabbit IgG sepharose beads. 5μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
“pro” stands for promoter region, while “NC2” stands for negative control which is negative loci at the promoter region.
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All lanes : Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557) at 1/2000 dilution
Lane 1 : NCCIT (Human pluripotent embryonic carcinoma epithelial cell) whole cell lysates
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 3 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates
Lane 4 : MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysates
Lane 5 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates
Lane 6 : Huh7 (Human hepatocellular carcinoma epithelial cell) whole cell lysates
Lane 7 : HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysates
Lane 8 : PANC-1 (Human pancreatic epithelioid carcinoma epithelial cell) whole cell lysates
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 39 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?
Exposure time: 3 secondsBlocking and diluting buffer: 5% NFDM/TBST
Oct4 is highly expressed in undifferentiated embryonic stem cells and cancer stem cell-like cells (PMID: 26013162, 21826175). ab181557 can’t detect the target band in undifferentiated cancer cell lines with low expression level of Oct4, such as HeLa, HEK-293, MDA-MB-231, HepG2, Huh7, HCT-116 and PANC-1 (PMID: 21975933, 29789579, 25625591, 26059097, 23928699, 27344963, 25837691, 29254202, 28854261, 27996162), even at the dilution of 1:200. -
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NCCIT (Human pluripotent embryonic carcinoma) cells (positive cell line) or NIH/3T3 (Mouse embyro fibroblast) cells (negative cell line) labeling Oct4 with ab181557 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Confocal image showing nuclear and weakly cytoplasmic staining on NCCIT cell line. Negative expression in NIH/3T3 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
-ve control 1: ab181557 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution. -
Chromatin was prepared from NCCIT (Human pluripotent embryonic carcinoma cell line) cells. ChIP was performed with 10^7 NCCIT cells and 8 µg of ab181557 [EPR17929]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.
Additional screenshots of mapped reads can be downloaded here. -
Immunocytochemistry/ Immunofluorescence - Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557)This image is courtesy of an Abreview submitted by Joanne Lacey.
ab181557 staining Oct4 in Human embryonic stem cells by ICC/IF (Immunocytochemistry/Immunofluorescence). Cells were fixed with formaldehyde , permeabilized with 0.1% Triton in PBS for 1 hour and blocked with 10% Serum for 1 hour at 25°C. Samples were incubated with primary antibody (1/200 in PBS with 0.1% Tween20) for 16 hours at 4°C. A monoclonal Goat Anti-rabbit Alexa Fluor® 594 was used as the secondary antibody at 1/200 dilution.
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Oct4 was immunoprecipitated from 1mg of NCCIT (Human pluripotent embryonic carcinoma) whole cell extract with ab181557 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab181557 at 1/10000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.
Lane 1: NCCIT whole cell extract 10 µg (Input). Lane 2: ab181557 IP in NCCIT whole cell extract. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab181557 in NCCIT whole cell extract.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.Exposure time: 3 minutes
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All lanes : Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557) at 1/1000 dilution
Lane 1 : NCCIT (Human pluripotent embryonic carcinoma) whole cell lysate
Lane 2 : F9 (Mouse embyro testicular cancer cell line) whole cell lysate
Lane 3 : NTERA-2 cl.D1 (Human malignant pluripotent embryonic carcinoma) whole cell lysate
Lane 4 : Mouse testis lysate
Lane 5 : Human hippocampus lysate
Lane 6 : Human cerebellum lysate
Lane 7 : Human testis lysate
Lane 8 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution
Predicted band size: 39 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?
Exposure time: 5 secondsBlocking/Dilution buffer: 5% NFDM/TBST.
Low levels of expression in adult tissues
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ab181557 staining OCT-4in the human cell line NCCIT (human pluripotent embryonal carcinoma) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/70. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody (Red).
Isotype control: Rabbit IgG monoclonal [EPR25A] ab172730 (Black).
Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue).
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557)
Immunohistochemical analysis of paraffin-embedded Human dysgerminoma of ovary tissue labeling Oct4 with ab181557 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear and weak cytoplasmic staining on cancer cells of Human dysgerminoma of ovary is observed. Counter stained with Hematoxylin.
Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557)
Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling Oct4 with ab181557 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Negative staining on Human breast cancer. Counter stained with Hematoxylin.
Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557)
Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling Oct4 with ab181557 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Negative staining on adult Human testis. Counter stained with Hematoxylin.
Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (106)
ab181557 has been referenced in 106 publications.
- Yan S et al. Extracellular magnetic labeling of biomimetic hydrogel-induced human mesenchymal stem cell spheroids with ferumoxytol for MRI tracking. Bioact Mater 19:418-428 (2023). PubMed: 35574059
- Zhang XZ et al. Folic Acid Rescues Valproic Acid-Induced Morphogenesis Inhibition in Neural Rosettes Derived From Human Pluripotent Stem Cells. Front Cell Neurosci 16:888152 (2022). PubMed: 35651759
- Barnada SM et al. Genomic features underlie the co-option of SVA transposons as cis-regulatory elements in human pluripotent stem cells. PLoS Genet 18:e1010225 (2022). PubMed: 35704668
- Chen X et al. VEGF-Loaded Heparinised Gelatine-Hydroxyapatite-Tricalcium Phosphate Scaffold Accelerates Bone Regeneration via Enhancing Osteogenesis-Angiogenesis Coupling. Front Bioeng Biotechnol 10:915181 (2022). PubMed: 35757798
- Gao X et al. Depletion of Fibroblast Growth Factor 12 Restrains the Viability, Stemness, and Motility of Colorectal Cancer. Biomed Res Int 2022:9948461 (2022). PubMed: 35860801