Key features and details
- Assay type: Semi-quantitative
- Detection method: Colorimetric
- Platform: Microplate reader
- Assay time: 3 hr 30 min
- Sample type: Nuclear Extracts
- Sensitivity: 500 ng/well
Product nameOct4 Transcription Factor Assay Kit (Colorimetric)
See all Oct4 kits
Sample typeNuclear Extracts
Assay time3h 30m
Species reactivityReacts with: Mouse, Rat, Human
Oct4 Transcription Factor Assay Kit (Colorimetric) (ab207224) is a high throughput assay to quantify Oct4 activation in nuclear extracts. This assay combines a quick ELISA format with a sensitive and specific non-radioactive assay for transcription factor activation.
A specific double stranded DNA sequence containing the Oct4 consensus binding site (5’ – ATTTGA A ATGCAAAT – 3’) has been immobilized onto a 96-well plate. Active Oct4 present in the nuclear extract specifically binds to the oligonucleotide. Oct4 is detected by a primary antibody that recognizes an epitope of Oct4 accessible only when the protein is activated and bound to its target DNA. An HRP-conjugated secondary antibody provides sensitive colorimetric readout that at OD 450 nm. This product detects human, mouse and rat Oct4.
Key performance and benefits:
- Assay time: 3.5 hours (cell extracts preparation not included).
- Detection limit: < 0.5 µg nuclear extract/well.
- Detection range: 0.5 – 5 µg nuclear extract/well.
Oct4 (Octamer Binding Transcription Factor 4) is part of the POU family, whose members contain a bipartite DNA-binding domain consisting of the POU-specific (POUS) and POU homeo-domain (POUHD). Connection of these subdomains by a flexible linker gives POU transcription factors the intrinsic ability to adopt several binding configurations on DNA. POU transcription factors were originally identified as proteins able to activate genes bearing cis-acting elements containing an octameric sequence within their promoter region. They bind to the consensus sequence ATGCAAAT.
The Oct4 gene has only been found in mammals. In mice, it is located on chromosome 17 and is separated into five exons, which encode a mRNA transcript of 1.5 kb. The human Oct4 is 87% identical to that of the mouse, and is located on chromosome 6. In humans, two transcripts are produced through alternative splicing, while mice produce only one transcript. Oct4 has also been found in other mammalian species including bovine and rat. Oct4 mRNA and protein are present in unfertilized oocytes and expression is abundant and uniform in all cells of the embryo through the morula stage (32-64 cells). After day 7.5, Oct4 is strictly confined to the primordial germ cells (PGCs), precursors of the gametes. Oct4 is also expressed in embryonic stem (ES) and embryonic carcinoma (EC) cells.
The first morphological indication of differentiation in mammalian embryogenesis is the formation of the trophectoderm (TE) at the early blastocyst stage. While TE cells are restricted to extra-embryonic cell lineages, inner cell mass (ICM) cells remain totipotent. During this phase, Oct4 is downregulated in cells that form the TE, while the cells in the ICM maintain a high level of Oct4. Consequently, a high level of Oct4 expression is thought to keep cells in a totipotent stage, whereas down-regulation is associated with differentiation. Several genes have been found to contain Oct4 binding sites; some positively and some negatively regulated. The α and β subunits of human chorionic gonadotrophin (HGC) are negatively regulated by Oct4 while platelet-derived growth factor (PDGF) α is activated by Oct4.
Storage instructionsPlease refer to protocols.
Components 1 x 96 tests 5 x 96 tests 10X Antibody Binding Buffer 1 x 2.2ml 1 x 11ml 10X Wash Buffer 1 x 22ml 1 x 110ml 96-well assay plate 1 unit 5 units Anti-rabbit HRP-conjugated IgG (0.25 μg/μL) 1 x 11µl 1 x 55µl Binding Buffer 1 x 10ml 1 x 50ml Developing Solution 1 x 11ml 1 x 55ml Dithiothreitol (DTT) (1 M) 1 x 100µl 1 x 500µl Herring sperm DNA (1 μg/μL) 1 x 100µl 1 x 500µl Lysis Buffer AM4 1 x 10ml 1 x 50ml Mutated oligonucleotide 1 x 100µl 1 x 500µl Oct-4 antibody 1 x 11µl 1 x 55µl P19 nuclear extract 1 x 40µl 1 x 200µl Plate sealer 1 unit 5 units Protease Inhibitor Cocktail 1 x 100µl 1 x 500µl Stop Solution 1 x 11ml 1 x 55ml Wild-type oligonucleotide 1 x 100µl 1 x 500µl
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.
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.
- Information by UniProt
- Octamer binding transcription factor 4
- Oct 3
ab207224 has not yet been referenced specifically in any publications.