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Synthetic peptide conjugated to KLH derived from within residues 300 to the C-terminus of Human Oct4.
(Peptide available as ab20650.)
Our Abpromise guarantee covers the use of ab19857 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IP||Use at an assay dependent concentration.|
|CHIPseq||Use at an assay dependent concentration. PubMed: 20526341|
|ICC/IF||Use a concentration of 1 - 5 µg/ml.|
|Flow Cyt||1/100. ab171870-Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.|
|IHC-Fr||1/200. (see Abreview)|
|IHC-P||1/250. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 43 kDa (predicted molecular weight: 39 kDa).Can be blocked with Human Oct4 peptide (ab20650).|
|ChIP||Use at an assay dependent concentration. PubMed: 18490265|
The image shows a colony of differentiating Human Embryonic Stem Cells double-stained with anti-Oct4 antibody ab19857 (green) and Sox17 antibody (red). The nuclei of Oct-4-positive undifferentiated hESCs stained bright green. Staining was restricted to the nuclei. Oct4-positive nuclei were Sox17-negative, and vice versa. This antibody can be used as a marker of Oct4-positive undifferentiated Human Embryonic Stem Cells.
Overlay histogram of Oct4 staining in mouse embryonic stem cells using ab19857 at 1:100 dilution. Purple histogram represents negative control (rabbit IgG); green line represents anti-Oct4 antibody (ab19857).
MEL-1 and MEL-2 Human Embryonic Stem Cell Lysates (ab27198 and ab27196), IOUD2 Oct4-expressing Mouse Embryonic Stem Cell Lysate (ab272020) and F9 Mouse Embryonic Carcinoma Cell Lysate (ab27193) all contain Oct4 protein. As expected, a band corresponding to Oct4 was detected in all four of the lysates by Westen Blot using anti-Oct4 antibody ab19857. Each of the Oct4 bands was specifically blocked using the immunising peptide of ab19857. The origin of the additional 65 kDa band detected in Lane 3 is unknown and may be a non-specific band that is recognised, in addition to Oct4, by ab19857 in mouse embryonic stem cells.
Mouse E6 - E6.5 embryos were sectioned and then fixed in paraformaldehyde. Antigen retrieval was performed using citric acid and the sections permeablized and blocked for 1 hour in serum. Embryos were stained with ab19857 (1:500) in 10% serum, 1% ovalbumin in PBSTween for 14 hours at 4C. They were then washed and stained with a goat anti-rabbit biotin conjugated antibody (1:000) and developed using ABC and then DAB for color. Staining is within the epiblast as expected.
Oct4 was immunoprecipitated using 0.5mg E14Tg2a whole cell extract, 5µg of Rabbit polyclonal to Oct4 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, E14Tg2a whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab19857.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 48kDa: Oct4.
ab19857 staining Oct4 in F9 cells treated with trans-retinoic acid (ab120728The cells were incubated at 37°C for 2 days in media containing different concentrations of ab120728ab96899) at 1:250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.
ICC/IF image of ab19857 stained Mouse Embryonic Stem cells. The cells were PFA fixed (4% PFA, 20 min) and incubated with the antibody (ab19857, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue).
The large nuclei of the Feeder cells can be seen in the image; ab19857 does not localise to these nuclei. ab19857 can be seen localising to the much smaller nuclei of the Mouse Embryonic Stem cells.
ab19857 staining OCT4 on genital ridges from E13.5 mouse embryos by immunohistochemistry. The tissue was paraformaldehyde fixed and permeabilized in 0.1% Triton prior to blocking in 1% BSA for 30 minutes at 20°C. The primary antibody was diluted 1:100 and incubated with the sample for 16 hours at 4°C. An Alexa Fluor 568 conjugated goat anti-rabbit antibody, diluted 1:300, was used as the secondary. Nuclear staining shown with DAPI.
ICC/IF image of ab19857 stained mouse embryonic stem cells. The cells were 4% formalin fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab19857, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1:00 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1:200 dilution for 1h. DAPI was used to stain the cell nuclei (blue).
Anti-Oct4 antibody ab19857 only detected a band corresponding to the expected size of Oct4 in human ES cell lysate. In mouse ES and EG cell lysates, ab19857 detected a band of approximately 55 kDa in addition to the expected 39 kDa Oct4 band.
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