Anti-Oct4 antibody - ChIP Grade (ab19857)
Overview
- Product nameAnti-Oct4 antibody - ChIP GradeSee all Oct4 primary antibodies ...
- DescriptionRabbit polyclonal to Oct4 - ChIP Grade
- SpecificityWB: Recognises a band of the expected size in mouse and human. Recognises an additional band in mouse but not in human.
- Tested applicationsIP, CHIPseq, ICC/IF, Flow Cyt, IHC-Fr, IHC-P, WB, ChIP more details
- Species reactivityReacts with: Mouse, Sheep, Human, Rhesus monkey
Predicted to work with: Rat - Immunogen
Synthetic peptide conjugated to KLH derived from within residues 300 to the C-terminus of Human Oct4.
(Peptide available as ab20650.)
- Positive controlHuman ES cell lysate
Properties
- FormLiquid
- Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
- Storage bufferPreservative: 0.02% Sodium Azide
Constituents: 1% BSA, PBS, pH 7.4
-
Concentration information loading... - PurityImmunogen affinity purified
- Clonality Polyclonal
- IsotypeIgG
-
Research Areas
Applications
Our Abpromise guarantee covers the use of ab19857 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Notes |
|---|---|
| IP | IP: Use at an assay dependent concentration. |
| CHIPseq | CHIPseq: Use at an assay dependent dilution. PubMed: 20526341 |
| ICC/IF | ICC/IF: Use a concentration of 1 - 5 µg/ml. |
| Flow Cyt | Flow Cyt: 1/100. |
| IHC-Fr | IHC-Fr: 1/200. (see Abreview) |
| IHC-P | IHC-P: 1/250. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol. |
| WB | WB: Use a concentration of 1 µg/ml. Detects a band of approximately 43 kDa (predicted molecular weight: 39 kDa).Can be blocked with Oct4 peptide (ab20650). |
| ChIP | ChIP: Use at an assay dependent dilution. PubMed: 18490265 |
Target
- FunctionTranscription 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 specificityExpressed 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 similaritiesBelongs to the POU transcription factor family. Class-5 subfamily.
Contains 1 homeobox DNA-binding domain.
Contains 1 POU-specific domain. - Developmental stageHighly expressed in undifferentiated embryonic stem cells and expression decreases gradually after embryoid body (EB) formation.
- DomainThe 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 localizationNucleus. Expressed in a diffuse and slightly punctuate pattern.
-
Database links
- Entrez Gene: 5460 Human
- Entrez Gene: 18999 Mouse
- Entrez Gene: 294562 Rat
- Omim: 164177 Human
- SwissProt: Q01860 Human
- SwissProt: P20263 Mouse
- Unigene: 249184 Human
- Unigene: 632482 Human
- Unigene: 646545 Human
- Unigene: 17031 Mouse
- Unigene: 161748 Rat
see all
Target information above from: UniProt accession
Q01860
The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010)
.
-
Alternative names
- MGC22487 antibodyOct 3 antibodyOct 4 antibody
- Oct-3 antibodyOct-4 antibodyOCT3 antibodyOct4 antibodyOctamer binding protein 3 antibodyOctamer binding protein 4 antibodyOctamer binding transcription factor 3 antibodyOctamer-binding protein 3 antibodyOctamer-binding protein 4 antibodyOctamer-binding transcription factor 3 antibodyOTF 3 antibodyOTF 4 antibodyOTF-3 antibodyOTF3 antibodyOTF4 antibodyPO5F1_HUMAN antibodyPOU class 5 homeobox 1 antibodyPOU domain class 5 transcription factor 1 antibodyPOU domain transcription factor OCT4 antibodyPOU domain, class 5, transcription factor 1 antibodyPOU type homeodomain containing DNA binding protein antibodyPOU5F1 antibody
see all
Anti-Oct4 antibody - ChIP Grade images
-
Immunocytochemistry/ Immunofluorescence - Anti-Oct4 antibody - ChIP Grade (ab19857)ab19857 staining Oct4 in F9 cells treated with trans-retinoic acid (ab120728The cells were incubated at 37°C for 2 days in media containing different concentrations of ab120728ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue. -
Immunofluorescence - Oct4 antibody - Embryonic Stem Cell Marker (ab19857)Ludovic Vallier, University of CambridgeThe image shows a colony of differentiating Human Embryonic Stem Cells double-stained with anti-Oct4 antibody ab19857 (green) and Sox17 antibody (red). The nuclei of Oct4-positive undifferentiated hESCs stained bright green. Staining was restricted to the nuclei. Oct4-positive nuclei were Sox17-negative, and vice versa. This antibody can be used as a marker of Oct4-positive undifferentiated Human Embryonic Stem Cells.The image shows a colony of differentiating Human Embryonic Stem Cells double-stained with anti-Oct4 antibody ab19857 (green) and Sox17 antibody (red). The nuclei of Oct4-positive undifferentiated hESCs stained bright green. Staining was restricted to the nuclei. Oct4-positive nuclei were Sox17-negative, and vice versa. This antibody can be used as a marker of Oct4-positive undifferentiated Human Embryonic Stem Cells. -
Western blotAll lanes : Anti-Oct4 antibody - ChIP Grade (ab19857) at 1 µg/ml
Lane 1 : MEL-1 (Human embryonic stem cell, male cell line) Whole Cell Lysate (ab27198)
Lane 2 : MEL-2 (Human embryonic stem cell, female cell line) Whole Cell Lysate (ab27196)
Lane 3 : IOUD2 (Mouse embryonic stem cell, selected for Oct4 expression cell line) Whole Cell Lysate (ab27202)
Lane 4 : F9 (Mouse embryonic carcinoma cell line) Whole Cell Lysate (ab27193)
Lane 5 : MEL-1 (Human embryonic stem cell, male cell line) Whole Cell Lysate (ab27198) withOct4 peptide (ab20650) at 1 µg/ml MEL-1 and MEL-2 Human Embryonic Stem Cell Lysates (ab27198 and ab27196), IOUD2 Oct4-expressing Mouse Embryonic Stem Cell Lysate (ab272020) and F9 Mouse Embryonic Carcinoma Cell Lysate (ab27193) all contain Oct4 protein. As expected, a band corresponding to Oct4 was detected in all four of the lysates by Westen Blot using anti-Oct4 antibody ab19857. Each of the Oct4 bands was specifically blocked using the immunising peptide of ab19857. The origin of the additional 65 kDa band detected in Lane 3 is unknown and may be a non-specific band that is recognised, in addition to Oct4, by ab19857 in mouse embryonic stem cells.
Lane 6 : MEL-2 (Human embryonic stem cell, female cell line) Whole Cell Lysate (ab27196) withOct4 peptide (ab20650) at 1 µg/ml
Lane 7 : IOUD2 (Mouse embryonic stem cell, selected for Oct4 expression cell line) Whole Cell Lysate (ab27202) withOct4 peptide (ab20650) at 1 µg/ml
Lane 8 : F9 (Mouse embryonic carcinoma cell line) Whole Cell Lysate (ab27193) withOct4 peptide (ab20650) at 1 µg/ml
Lysates/proteins at 10 µl per lane.
Observed band size : 48 kDa (why is the actual band size different from the predicted?)
Additional bands at : 65 kDa. We are unsure as to the identity of these extra bands. -
Immunocytochemistry/ Immunofluorescence - Oct4 antibody - Embryonic Stem Cell Marker (ab19857)ICC/IF image of ab19857 stained mouse embryonic stem cells. The cells were 4% formalin fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab19857, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue). -
Immunocytochemistry/ Immunofluorescence - Oct4 antibody - Embryonic Stem Cell Marker (ab19857)ICC/IF image of ab19857 stained Mouse Embryonic Stem cells. The cells were PFA fixed (4% PFA, 20 min) and incubated with the antibody (ab19857, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue).
The large nuclei of the Feeder cells can be seen in the image; ab19857 does not localise to these nuclei. ab19857 can be seen localising to the much smaller nuclei of the Mouse Embryonic Stem cells.
-
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Oct4 antibody - Embryonic Stem Cell Marker (ab19857)Mouse E6 - E6.5 embryos were sectioned and then fixed in paraformaldehyde. Antigen retrieval was performed using citric acid and the sections permeablized and blocked for 1 hour in serum. Embryos were stained with ab19857 (1/2500) in 10% serum, 1% ovalbumin in PBSTween for 14 hours at 4C. They were then washed and stained with a goat anti-rabbit biotin conjugated antibody (1/2000) and developed using ABC and then DAB for color. Staining is within the epiblast as expected.
-
Flow Cytometry - Anti-Oct4 antibody - ChIP Grade (ab19857)This image is taken from an abreview by Kate HawkinsOverlay histogram of Oct4 staining in mouse embryonic stem cells using ab19857 at 1:100 dilution. Purple histogram represents negative control (rabbit IgG); green line represents anti-Oct4 antibody (ab19857). -
Immunoprecipitation - Anti-Oct4 antibody - ChIP Grade (ab19857)Oct4 was immunoprecipitated using 0.5mg E14Tg2a whole cell extract, 5µg of Rabbit polyclonal to Oct4 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, E14Tg2a whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab19857.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 48kDa: Oct4. -
Immunohistochemistry (Frozen sections) - Anti-Oct4 antibody - ChIP Grade (ab19857)This image is courtesy of an abreview from Rachel Amouroux.ab19857 staining OCT4 on genital ridges from E13.5 mouse embryos by immunohistochemistry. The tissue was paraformaldehyde fixed and permeabilized in 0.1% Triton prior to blocking in 1% BSA for 30 minutes at 20°C. The primary antibody was diluted 1/100 and incubated with the sample for 16 hours at 4°C. An Alexa Fluor 568 conjugated goat anti-rabbit antibody, diluted 1/300, was used as the secondary. Nuclear staining shown with DAPI. -
Western blotAll lanes : Anti-Oct4 antibody - ChIP Grade (ab19857) at 1 µg/ml
Lane 1 : Human embryonic stem cell lysate
Lane 2 : Mouse embryonic stem cell lysate
Lane 3 : Mouse embryonic germ cell lysate
Lane 4 : Human embryonic stem cell lysate withOct4 peptide (ab20650) at 1 µg/ml Anti-Oct4 antibody ab19857 only detected a band corresponding to the expected size of Oct4 in human ES cell lysate. In mouse ES and EG cell lysates, ab19857 detected a band of approximately 55 kDa in addition to the expected 39 kDa Oct4 band.
Lane 5 : Mouse embryonic stem cell lysate withOct4 peptide (ab20650) at 1 µg/ml
Lane 6 : Mouse embryonic germ cell lysate withOct4 peptide (ab20650) at 1 µg/ml
Lysates/proteins at 20 µg per lane.
Predicted band size : 39 kDa
Observed band size : 43 kDa (why is the actual band size different from the predicted?)
Additional bands at : 55 kDa (possible glycosylated form,cross reactivity).
References for Anti-Oct4 antibody - ChIP Grade (ab19857)
This product has been referenced in:
- Dietrich N et al. REST-mediated recruitment of polycomb repressor complexes in mammalian cells. PLoS Genet 8:e1002494 (2012). WB ; Mouse . Read more (PubMed: 22396653) »
- Bhartiya D et al. Very Small Embryonic- like Stem Cells with Maximum Regenerative Potential get Discarded during Cord Blood Banking and Bone Marrow Processing for Autologous Stem Cell Therapy. Stem Cells Dev : (2011). ICC ; Human . Read more (PubMed: 21780911) »
