Anti-Dnmt1 antibody [EPR3521(2)]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Dnmt1 antibody [EPR3521(2)] (AB134148)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Dnmt1 (red) with unpurified ab134148 at a 1/20 dilution (10ug/mL). Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (Black) (ab172730). Blue (unlabeled control) - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt1 antibody [EPR3521(2)] (AB134148)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling Dnmt1 with purified ab134148 at 1/700. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Dnmt1 antibody [EPR3521(2)] (AB134148)

ab134148 staining Dnmt1 in wild-type HAP1 cells (top panel) and DNMT1 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 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 with ab134148 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt1 antibody [EPR3521(2)] (AB134148)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human stomach tissue labelling Dnmt1 with unpurified ab134148 at a dilution of 1/250.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt1 antibody [EPR3521(2)] (AB134148)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling Dnmt1 with unpurified ab134148 at a dilution of 1/250.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• WB

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Western blot - Anti-Dnmt1 antibody [EPR3521(2)] (AB134148)

All lanes:

Western blot - Anti-Dnmt1 antibody [EPR3521(2)] (ab134148) at 1/1000 dilution

Lane 1:

Jurkat cell lysates at 10 µg

Lane 2:

293T cell lysates at 10 µg

Lane 3:

HeLa cell lysates at 10 µg

Lane 4:

HuT-78 cell lysates at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 183 kDa

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• WB

Lab

Western blot - Anti-Dnmt1 antibody [EPR3521(2)] (AB134148)

Blocking and dilution buffer : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Dnmt1 antibody [EPR3521(2)] (ab134148) at 1/5000 dilution

All lanes:

Jurkat whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 183 kDa

Observed band size: 183 kDa

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• WB

Lab

Western blot - Anti-Dnmt1 antibody [EPR3521(2)] (AB134148)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : DNMT1 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lane 4 : HEK293 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab134148 observed at 183 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab134148 was shown to recognize DNMT1 when DNMT1 knockout samples were used, along with additional cross-reactive bands. Wild-type and DNMT1 knockout samples were subjected to SDS-PAGE. ab134148 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 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 [EPR3521(2)] (ab134148)

Predicted band size: 183 kDa

false

• WB

Lab

Western blot - Anti-Dnmt1 antibody [EPR3521(2)] (AB134148)

Blocking and dilution buffer : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Dnmt1 antibody [EPR3521(2)] (ab134148) at 1/5000 dilution

All lanes:

293T whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 183 kDa

Observed band size: 183 kDa

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