Anti-Dnmt1 antibody [EPR3522]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Dnmt1 antibody [EPR3522] (AB92314)

Immunofluorescence staining of Jurkat cells with purified ab92314 at a working dilution of 1/2000, counter-stained with DAPI. The secondary antibody was an Alexa Fluor^® 488 conjugated goat anti-rabbit (ab150077), used at a dilution of 1/1000. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative control is shown in bottom right hand panel - for the negative control, PBS was used instead of the primary antibody.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Dnmt1 antibody [EPR3522] (AB92314)

Overlay histogram showing HeLa cells stained with ab92314 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab92314, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt1 antibody [EPR3522] (AB92314)

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling Dnmt1 with ab92314 at 1/200 (3.55 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human placenta. The section was incubated with ab92314 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt1 antibody [EPR3522] (AB92314)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling Dnmt1 with ab92314 at 1/200 (3.55 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human tonsil. The section was incubated with ab92314 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IP

Lab

Immunoprecipitation - Anti-Dnmt1 antibody [EPR3522] (AB92314)

Dnmt1 was immunoprecipitated from 0.35 mg Hut-78 (Human Sezary syndrome cutaneous T lymphocyte) whole cell lysate 10 μg with 92314 at 1/50 dilution (2μg). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : Hut-78 (Human Sezary syndrome cutaneous T lymphocyte) whole cell lysate 10 μg

Lane 2 : ab92314 IP in Hut-78 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab92314 in Hut-78 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Dnmt1 antibody [EPR3522] (ab92314)

Predicted band size: 183 kDa

Observed band size: 183 kDa

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• WB

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Western blot - Anti-Dnmt1 antibody [EPR3522] (AB92314)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : DNMT1 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HEK293 whole cell lysate (20 μg)
Lane 4 : HeLa whole cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab92314 observed at 170 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab92314 was shown to specifically react with DNMT1 when DNMT1 knockout samples were used. Wild-type and DNMT1 knockout samples were subjected to SDS-PAGE. ab92314 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1000 dilution and 1/10000 dilution respectively. Blots were developed with 800CW Goat anti Rabbit and 680CW Goat anti Mouse secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Dnmt1 antibody [EPR3522] (ab92314)

Predicted band size: 183 kDa

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• WB

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Western blot - Anti-Dnmt1 antibody [EPR3522] (AB92314)

All lanes:

Western blot - Anti-Dnmt1 antibody [EPR3522] (ab92314) at 1/1000 dilution

Lane 1:

HuT-78 cell lysate at 10 µg

Lane 2:

Jurkat cell lysate at 10 µg

Lane 3:

293T cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 183 kDa

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