Anti-Oct4 antibody

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Oct4 antibody (AB19857)

ab19857 staining Oct4 in mES cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween 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 ab19857 at 1μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-Oct4 antibody (AB19857)

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.

This image is courtesy of Ludovic Vallier, University of Cambridge

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Oct4 antibody (AB19857)

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-iT™ 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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Oct4 antibody (AB19857)

ab19857 staining Oct4 in F9 cells treated with trans-retinoic acid (ab120728) The cells were incubated at 37°C for 2 days in media containing different concentrations of ab120728 (trans-retinoic acid) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab19857 (1 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight^® 488 anti-rabbit polyclonal antibody (ab96899) at 1 : 250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

• IP

Unknown

Immunoprecipitation - Anti-Oct4 antibody (AB19857)

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 70^oC; 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.

All lanes:

Immunoprecipitation - Anti-Oct4 antibody (ab19857)

Predicted band size: 38 kDa

false

• WB

Lab

Western blot - Anti-Oct4 antibody (AB19857)

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.

All lanes:

Western blot - Anti-Oct4 antibody (ab19857) at 1 µg/mL

Lane 1:

Human embryonic stem cell lysate at 20 µg

Lane 2:

Mouse embryonic stem cell lysate at 20 µg

Lane 3:

Mouse embryonic germ cell lysate at 20 µg

Lane 4:

Human embryonic stem cell lysate at 20 µg with Human Oct4 peptide (<a href='/en-us/products/proteins-peptides/human-oct4-peptide-ab20650'>ab20650</a>)

Lane 5:

Mouse embryonic stem cell lysate at 20 µg with Human Oct4 peptide (<a href='/en-us/products/proteins-peptides/human-oct4-peptide-ab20650'>ab20650</a>)

Lane 6:

Mouse embryonic germ cell lysate at 20 µg with Human Oct4 peptide (<a href='/en-us/products/proteins-peptides/human-oct4-peptide-ab20650'>ab20650</a>)

Predicted band size: 38 kDa

Observed band size: 43 kDa,55 kDa

false

• WB

Unknown

Western blot - 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 (ab27202) 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.

All lanes:

Western blot - Anti-Oct4 antibody (ab19857) at 1 µg/mL

Lane 1:

MEL-1 (Human embryonic stem cell, male cell line) Whole Cell Lysate (<a href='/en-us/products/unavailable/mel-1-human-embryonic-stem-cell-male-cell-line-whole-cell-lysate-ab27198'>ab27198</a>) at 10 µL

Lane 2:

MEL-2 (Human embryonic stem cell, female cell line) Whole Cell Lysate (<a href='/en-us/products/unavailable/mel-2-human-embryonic-stem-cell-female-cell-line-whole-cell-lysate-ab27196'>ab27196</a>) at 10 µL

Lane 3:

IOUD2 (Mouse embryonic stem cell, selected for Oct4 expression cell line) Whole Cell Lysate (<a href='/en-us/products/unavailable/ioud2-mouse-embryonic-stem-cell-selected-for-oct4-expression-cell-line-whole-cell-lysate-ab27202'>ab27202</a>) at 10 µL

Lane 4:

F9 (Mouse embryonic carcinoma cell line) Whole Cell Lysate (<a href='/en-us/products/unavailable/f9-mouse-embryonic-carcinoma-cell-line-whole-cell-lysate-ab27193'>ab27193</a>) at 10 µL

Lane 5:

MEL-1 (Human embryonic stem cell, male cell line) Whole Cell Lysate (<a href='/en-us/products/unavailable/mel-1-human-embryonic-stem-cell-male-cell-line-whole-cell-lysate-ab27198'>ab27198</a>) at 10 µL with Human Oct4 peptide (<a href='/en-us/products/proteins-peptides/human-oct4-peptide-ab20650'>ab20650</a>)

Lane 6:

MEL-2 (Human embryonic stem cell, female cell line) Whole Cell Lysate (<a href='/en-us/products/unavailable/mel-2-human-embryonic-stem-cell-female-cell-line-whole-cell-lysate-ab27196'>ab27196</a>) at 10 µL with Human Oct4 peptide (<a href='/en-us/products/proteins-peptides/human-oct4-peptide-ab20650'>ab20650</a>)

Lane 7:

IOUD2 (Mouse embryonic stem cell, selected for Oct4 expression cell line) Whole Cell Lysate (<a href='/en-us/products/unavailable/ioud2-mouse-embryonic-stem-cell-selected-for-oct4-expression-cell-line-whole-cell-lysate-ab27202'>ab27202</a>) at 10 µL with Human Oct4 peptide (<a href='/en-us/products/proteins-peptides/human-oct4-peptide-ab20650'>ab20650</a>)

Lane 8:

F9 (Mouse embryonic carcinoma cell line) Whole Cell Lysate (<a href='/en-us/products/unavailable/f9-mouse-embryonic-carcinoma-cell-line-whole-cell-lysate-ab27193'>ab27193</a>) at 10 µL with Human Oct4 peptide (<a href='/en-us/products/proteins-peptides/human-oct4-peptide-ab20650'>ab20650</a>)

Predicted band size: 38 kDa

Observed band size: 48 kDa,65 kDa

false