Anti-Oct4 antibody [EPR17929] - ChIP Grade

13 product images

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  Immunocytochemistry/ Immunofluorescence - Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557)

  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) (Goat Anti-Rabbit IgG H&L (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 Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed 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 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
  -ve control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

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

  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.

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

  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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab181557 in NCCIT whole cell extract.
  Blocking and dilution buffer and concentration: 5% NFDM/TBST.

  Exposure time: 3 minutes

  All lanes: Immunoprecipitation - Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557)

  Predicted band size: 38 kDa

  Observed band size: 45 kDa

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

  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.

  All lanes: Western blot - Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557) at 1/2000 dilution

  Lane 1: NCCIT (Human pluripotent embryonic carcinoma epithelial cell) whole cell lysates at 20 µg

  Lane 2: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

  Lane 3: HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates at 20 µg

  Lane 4: MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 20 µg

  Lane 5: HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates at 20 µg

  Lane 6: Huh7 (Human hepatocellular carcinoma epithelial cell) whole cell lysates at 20 µg

  Lane 7: HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysates at 20 µg

  Lane 8: PANC-1 (Human pancreatic epithelioid carcinoma epithelial cell) whole cell lysates at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 38 kDa

  Observed band size: 45 kDa

  Exposure time: 3s

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  Flow Cytometry (Intracellular) - Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557)

  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] Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 (Black).

  Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue).

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  This image is courtesy of a customer review submitted by Joanne Lacey.

  Immunocytochemistry/ Immunofluorescence - Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557)

  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.

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

  Blocking/Dilution buffer: 5% NFDM/TBST.

  Low levels of expression in adult tissues

  All lanes: Western blot - Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557) at 1/1000 dilution

  Lane 1: NCCIT (Human pluripotent embryonic carcinoma) whole cell lysate at 10 µg

  Lane 2: F9 (Mouse embyro testicular cancer cell line) whole cell lysate at 10 µg

  Lane 3: NTERA-2 cl.D1 (Human malignant pluripotent embryonic carcinoma) whole cell lysate at 10 µg

  Lane 4: Mouse testis lysate at 10 µg

  Lane 5: Human hippocampus lysate at 10 µg

  Lane 6: Human cerebellum lysate at 10 µg

  Lane 7: Human testis lysate at 10 µg

  Lane 8: NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate at 10 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

  Predicted band size: 38 kDa

  Observed band size: 45 kDa

  Exposure time: 5s

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557)

  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) (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) (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.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557)

  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) (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) (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.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557)

  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) (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) (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.

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  Flow Cytometry (Intracellular) - 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.

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

  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.

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  Image collected and cropped by CiteAb under a CC-BY license from the publication

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

  Oct4 western blot using anti-Oct4 antibody [EPR17929] - ChIP Grade ab181557. Publication image and figure legend from Kim, H. S., Yoon, J. W., et al., 2017, Sci Rep, PubMed 29062050.

  
  

  ab181557 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab181557 please see the product overview.

  Characterization of cardiomyocytes differentiated from H9 hESCs. (a) Bright field images of H9 cells at day 0 (left panel) and differentiating cells at day 30 (right panel). Scale bar = 100 μm (b) Differentiated cardiomyocytes (hESC-CMs) at day 30 were subjected to immunofluorescent staining with antibodies against α-SA (red), cTnT (green), or MLC2a (yellow). Nuclei were stained with DAPI, and the merged images are shown. Scale bar = 20 μm. (c) Protein expression in undifferentiated H9 cells, hESC-CMs, and adult human left ventricular tissue (adult hLV) was assessed by Western blot analysis with antibodies against pluripotency markers (OCT4 and NANOG) and cardiomyocyte markers (α-SA, cTnT, MLC2a, MLC2v, SERCA2a and Cx43). (d) The mRNA levels of α-MHC and β-MHC in hESC-CMs and adult hLV were accessed by quantitative RT-PCR and the ratio of β-MHC/α-MHC mRNA levels was determined (n = 3). *P < 0.05. (e) Gene expression in hESC-CMs at day 30 and hESCs was accessed by quantitative RT-PCR with cardiomyocyte markers (GATA4, GATA6, and cTnT) and pluripotency markers (OCT4 and NANOG) n = 3. *P < 0.05. (f) Flow cytometry analysis of hESC-CMs at day 30 with antibodies against cardiomyocyte markers (cTnT, MLC2a, and α-SA) and pluripotency markers (TRA-1-60 and SSEA3) is shown.