Total lysates (15 µg protein) from NTERA2 (lane 1), HeLa (lane 2) and NF-1 (lane 3) cells were resolved by electrophoresis (4-12% Bis-Tris gel), transferred to nitrocellulose, and probed with 1:1000 diluted (0.5 µg/mL) Purified anti-Oct4(Oct3) Antibody, clone 3A2A20 (upper), or 1:2000 diluted anti-GAPDH Antibody (lower). Proteins were visualized by chemiluminescence detection using a 1:3000 diluted goat anti-mouse-IgG secondary antibody conjugated to HRP for the anti-Oct4 Antibody or 1:3000 diluted Donkey anti-Rabbit-IgG secondary antibody conjugated to HRP for anti-GAPDH Antibody. Lane M: Molecular weight ladder.
Total lysates (15 µg protein) from NTERA2 (lane 1), HeLa (lane 2) and NF-1 (lane 3) cells were resolved by electrophoresis (4-12% Bis-Tris gel), transferred to nitrocellulose, and probed with 1:1000 diluted (0.5 µg/mL) Purified anti-Oct4(Oct3) Antibody, clone 3A2A20 (upper), or 1:2000 diluted anti-GAPDH Antibody (lower). Proteins were visualized by chemiluminescence detection using a 1:3000 diluted goat anti-mouse-IgG secondary antibody conjugated to HRP for the anti-Oct4 Antibody or 1:3000 diluted Donkey anti-Rabbit-IgG secondary antibody conjugated to HRP for anti-GAPDH Antibody. Lane M: Molecular weight ladder.
NTERA-2 cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes, permeabilized with 0.5% Triton X-100 for 5 minutes, and blocked with 5% FBS for 30 minutes. Then the cells were intracellularly stained with 2.5 µg/mL anti-Oct4 (Oct3) (3A2A20) and followed by DyLight™ 594 goat anti-mouse IgG (red) for 1 hour at room temperature. Actin filaments were labeled with Alexa Fluor® 488 Phalloidin (green). Nuclei were counterstained with DAPI (blue). The image was captured with a 40X objective.
Chromatin Immunoprecipitations (ChIP) were performed with cross-linked chromatin samples from 4 X 106 NCCIT cells with either A) 1:50 dilution of Go-ChIP-Grade™ Purified anti-Oct4 (Oct3) Antibody (clone 3A2A20, Cat. No. 653713) or B) equal amount of Purified Mouse IgG2b, κ isotype control antibody (clone MPC-11, Cat. No. 400301) by using Go-ChIP-Grade™ Protein G Enzymatic Kit (Cat. No. 699904). The enriched DNA was purified and quantified by real-time qPCR using primers targeting human SOX2 gene region or α-Satellite repeats. The amount of immunoprecipitated DNA in each sample is represented as signal relative to total amount of input chromatin.
Chromatin Immunoprecipitations (ChIP) were performed with cross-linked chromatin samples from 4 X 106 NCCIT cells with either A) 1:50 dilution of Go-ChIP-Grade™ Purified anti-Oct4 (Oct3) Antibody (clone 3A2A20, Cat. No. 653713) or B) equal amount of Purified Mouse IgG2b, κ isotype control antibody (clone MPC-11, Cat. No. 400301) by using Go-ChIP-Grade™ Protein G Enzymatic Kit (Cat. No. 699904). The enriched DNA was purified and quantified by real-time qPCR using primers targeting human OCT4 gene region or α-Satellite repeats. The amount of immunoprecipitated DNA in each sample is represented as signal relative to total amount of input chromatin.
Oct4 (Octamer binding transcription factor 4) is an important marker of the undifferentiated state and central regulator of pluripotency in ES cells. When embryonic stem cells are triggered to differentiate, Oct4 is downregulated, thus providing a model for the early events linked to somatic differentiation in the developing embryo. Oct4 is a nuclear protein, with a predicted molecular weight of 39 kD.
This antibody is provided in phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide. Previous lots of this product may have been formulated with 0.1% or 0.05% NaN3 instead of 0.09% NaN3. For further information please contact BioLegend Technical Support or Customer Service.
Concentration
0.5 mg/ml
Storage & Handling
Upon receipt, store undiluted between 2°C and 8°C.
Each lot of this antibody is quality control tested by Western blotting. Western blotting, suggested working dilution(s): Use 0.5 µg/ml antibody dilution buffer for each mini-gel. For immunocytochemistry, a concentration of 2.5 µg/ml is recommended. For ChIP applications, the suggested use of this reagent is 1:50 to 1:100 by volume. It is recommended that the reagent be titrated for optimal performance for each application.
Belongs to the POU transcription factor family, predicted molecular weight of 39 kD
Distribution
Nuclear protein, expressed in undifferentiated embryonal carcinoma (EC) cells, embryonic stem (ES) cells and embryonic germ (EG) cells
Function
Transcription factor that binds to the octamer motif (5'-ATTTGCAT-3'). Oct4 is a prime candidate for an early developmental control gene. It also is an important marker of the undifferentiated state and central regulator of pluripotency in ES cells.
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