Recombinant Anti-Oct4 antibody [EPR17980] - BSA and Azide free (ab240358)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/µL, 2 x 10^5 NCCIT (Human pluripotent embryonic carcinoma cell line) cells and 5 µg of ab200834 [EPR17980]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. The ChIP data was conducted on chromatin prepared from NCCIT cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 NCCIT cells and 8 µg of ab200834. 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.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab200834).

ab200834 staining Oct4 in F9 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab200834 at a 5μg/ml concentration and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1μg/ml concentration, followed by a further incubation at room temperature for 1h with an anti-rabbit AlexaFluor® 488 (ab150081) at 2 μg/ml (shown in green) and an anti-mouse AlexaFluor® 594 (ab150120) at 2 μg/ml (shown in pseudocolor red).  Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab200834).

Immunocytochemistry/Immunofluorescence analysis of F9 (mouse embryonal carcinoma) labelling Oct4 with purified ab200834 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Ab150077). Nuclei counterstained with DAPI (blue).

Control: PBS only

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab200834).

Oct4 was immunoprecipitated from 1mg of F9 (Mouse embyro testicular cancer cell line) whole cell lysate with ab200834 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab200834 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: F9 whole cell lysate 10 µg (Input). Lane 2: ab200834 IP in F9 whole cell lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab200834 in F9 whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 5 seconds

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab200834).

Intracellular flow cytometric analysis of 2% paraformaldehyde-fixed F9 (Mouse embyro testicular cancer cell line) cells labeling Oct4 with ab200834 at 1/60 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab200834). 

Immunohistochemical analysis of paraffin-embedded Human cerebral cortex tissue labeling Oct4 with ab200834 at 1/500 dilution, followed Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Human cerebral cortex tissue is a negative control for Oct4. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab200834).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human spermatocytoma tissue labeling Oct4 with ab200834 at 1/500 dilution, followed Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear and weakly cytoplasmic staining on Human spermatocytoma tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab200834).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human dysgerminoma of ovary tissue labeling Oct4 with ab200834 at 1/500 dilution, followed Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear and weakly cytoplasmic staining on Human dysgerminoma of ovary tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab200834).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Chromatin was prepared from NCCIT (Human pluripotent embryonic carcinoma cell line) cells. ChIP was performed with 10^7 NCCIT cells and 8 µg of ab200834 [EPR17980]. 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab200834).