Recombinant Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557)

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.

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.

Blocking 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.

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.

See Abreview

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

Blocking/Dilution buffer: 5% NFDM/TBST.

Low levels of expression in adult tissues

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). 

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.

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.

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.