重组Anti-Dnmt1抗体[RM1192] (ab317845)

Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of TBS.

Lysates at 20 µg per lane.

The samples were run on a Bis-Tris gel under reducing conditions.

Western blot: Anti-Dnmt1 antibody (ab317845) staining at 1/1000 dilution and Anti-Dnmt1 antibody (ab19905) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5](ab8245) loading control staining at 1/20000 dilution, shown in magenta.

In Western blot, ab317845 was shown to bind specifically to Dnmt1. Target of interest was observed at 183-184 kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in Dnmt1 knockout cell line (lane 2). To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 31426844).

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 31426844).

The lysates were freshly made and used for Western Blotting immediately to minimize protein degradation.

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 31426844).

The lysates were freshly made and used for Western Blotting immediately to minimize protein degradation.

Exposure time: Lane 1-2: 10 seconds Lane 2: 70 seconds

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 31426844).

The lysates were suggested freshly made and used for Western Blotting immediately to minimize protein degradation.

Exposure time: Lane 1:180 seconds Lane 2: 70 seconds

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling Dnmt1 with ab317845 at 1/500 (1.062 ug/ml) dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer) (ab214880).

Positve staining on human tonsil. The section was incubated with ab317845 at 4°C overnight.

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer) (ab214880).

Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0)

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling Dnmt1 with ab317845 at 1/100 (5.31 ug/ml) dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer) (ab214880).

Positive staining on mouse testis. The section was incubated with ab317845 at 4°C overnight.

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer) (ab214880).

Heat mediated antigen retrieval was performed using ab93678 (citrate buffer, pH 6.0)

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling Dnmt1 with ab317845 at 1/100 (5.31 ug/ml) dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer) (ab214880).

Positive staining on rat testis. The section was incubated with ab317845 at 4°C overnight.

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer) (ab214880).

Heat mediated antigen retrieval was performed using ab93678 (citrate buffer, pH 6.0)

Immunohistochemical analysis of paraffin-embedded A Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) cell pellet. B Dnmt1 knockout HAP1 cell pellet. tissue labeling Dnmt1 with ab317845 at 1/500 (1.062 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond ™ Polymer Refine Detection).

Positive staining on (A) wild-type HAP1 cell pellet, no staining on (B) Dnmt1 knockout HAP1 cell pellet. The section was incubated with ab317845 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND ® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a 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

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized parental HAP1(HOWT01) (human chronic myelogenous leukemia near-haploid cell) cells labelling Dnmt1 with ab317845 at 1/500 (1.062 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 wildtype 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 2.5ug/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.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Neuro-2a (mouse neuroblastoma neuroblast) cells labelling Dnmt1 with ab317845 at 1/500 (1.062 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 neuro-2a cell line (shown in green). 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 2.5ug/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.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Parental HAP1 (HOWT01) (human chronic myelogenous leukemia near-haploid cell) (Left) / DNMT1 knockout HAP1(HO010163) (Right) cells labelling Dnmt1 with ab317845 at 1/5000 dilution (0.1ug) (Magenta) 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-Rabbit IgG (Alexa Fluor ® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Dnmt1 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab317845 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317845 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 2: ab317845 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab317845 in HeLa whole cell lysate

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

Exposure time: 8 seconds.

The bands beneath the target band are likely to be degraded target fragments.