Recombinant 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.

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.

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.

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.