Anti-Dnmt1 antibody [EPR28422-79] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Dnmt1 antibody [EPR28422-79] - BSA and Azide free (AB320818)

This data was developed using ab320817, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized parental HAP1 (HOWT01) (human chronic myelogenous leukemia near-haploid cell, right) / DNMT1 KO HAP1(HO010163) (Left) cells labelling Dnmt1 with ab320817 at 1/50000 dilution (0.001ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Mouse IgG (Alexa Fluor® 488, ab150113) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Dnmt1 antibody [EPR28422-79] - BSA and Azide free (AB320818)

This data was developed using ab320817, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized DNMT1 KO HAP1 (DNMT1 knockout human chronic myelogenous leukemia near-haploid cell) cells labelling Dnmt1 with ab320817 at 1/4000 (0.125 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing nuclear staining in HAP1 cell line (shown in green) and no staining in DNMT1 KO HAP1 cell line. The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• ChIP-seq

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ChIP-sequencing - Anti-Dnmt1 antibody [EPR28422-79] - BSA and Azide free (AB320818)

This data was developed using ab320817, the same antibody clone in a different buffer formulation.

Chromatin was prepared from HepG2 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 µg of ab320817 [EPR28422-79]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 60 million reads. The Input control is also shown.

• IP

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Immunoprecipitation - Anti-Dnmt1 antibody [EPR28422-79] - BSA and Azide free (AB320818)

This data was developed using ab320817, the same antibody clone in a different buffer formulation.

Dnmt1 was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab320817 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab320817 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 2 : ab320817 IP in HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab320817 in HepG2 whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-Dnmt1 antibody [EPR28422-79] (<a href='/ko/products/primary-antibodies/dnmt1-antibody-epr28422-79-ab320817'>ab320817</a>) at 1/30 dilution

All lanes:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/ko/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 180s

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-Dnmt1 antibody [EPR28422-79] - BSA and Azide free (AB320818)

This data was developed using ab320817, the same antibody clone in a different buffer formulation.

Chromatin was prepared from HepG2 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 µg of ab320817 [EPR28422-79]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 60 million reads. The Input control is also shown.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-Dnmt1 antibody [EPR28422-79] - BSA and Azide free (AB320818)

This data was developed using ab320817, the same antibody clone in a different buffer formulation.

Chromatin was prepared from HepG2 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 µg of ab320817 [EPR28422-79]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 60 million reads. The Input control is also shown.

• WB

Supplier Data

Western blot - Anti-Dnmt1 antibody [EPR28422-79] - BSA and Azide free (AB320818)

This data was developed using ab320817, the same antibody clone in a different buffer formulation.

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

In Western blot, ab320817 was shown to bind specifically to Dnmt1. Target of interest was observed at 183 kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in Dnmt1 knockout cell line (lane 2).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Dnmt1 antibody [EPR28422-79] (<a href='/ko/products/primary-antibodies/dnmt1-antibody-epr28422-79-ab320817'>ab320817</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

Lane 2:

Dnmt knockout HAP1 whole cell lysate at 20 µg

Lane 3:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 4:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 183 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Dnmt1 antibody [EPR28422-79] - BSA and Azide free (AB320818)

This data was developed using ab320817, the same antibody clone in a different buffer formulation.

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

All lanes:

Western blot - Anti-Dnmt1 antibody [EPR28422-79] (<a href='/ko/products/primary-antibodies/dnmt1-antibody-epr28422-79-ab320817'>ab320817</a>) at 1/1000 dilution

Lane 1:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate at 40 µg

Lane 2:

Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate at 40 µg

Lane 3:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 40 µg

Lane 4:

Ramos (human Burkitts lymphoma B lymphocyte) whole cell lysate at 40 µg

Lane 5:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 183 kDa

false

Exposure time: 180s