Recombinant Anti-ADAR1 antibody [EPR25431-60] (ab307585)

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

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

Exposure time: 70 seconds

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

Performed under reducing conditions.

In Western blot, ab307585 was shown to bind specifically to ADAR1. A band was observed at 150 kDa in wild-type HEK-293T cell lysates whereas no signal observed at this size in ADAR1 knockout cell line ab266846 (knockout cell lysate ab257131).

Exposure time: 59 seconds

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

Exposure time: 37 seconds

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

Exposure time: 158 seconds

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling ADAR1 with ab307585 at 1/500 (1.08 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 increased nuclear and cytoplasmic staining in HeLa cells treated with IFN alpha 1 (human) (10 ng/ml) for 16 hours. 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 (Red). 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 ADAR KO HEK293T (ab266846) cells labelling ADAR1 with ab307585 at 1/500 (1.08 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 increased nuclear and cytoplasmic staining in parental HEK293T cells treated with IFN alpha 1 (human) (10 ng/ml) for 16 hours, and no staining in treated ADAR KO HEK293T cells. 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 (Red). 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 Wild-type 293T (human embryonic kidney epithelial cell, Right) / ADAR1 knockout 293T (Left) cells labelling ADAR1 with ab307585 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

ADAR1 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate 10 ug with ab307585 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307585 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 10 ug

Lane 2: ab307585 IP in HeLa whole cell lysate

Lane 3:Rabbit monoclonal IgG (ab172730) instead of ab307585 in HeLa whole cell lysate

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

Exposure time: Lanes 1-3: 5 seconds (left), Lanes 1-3: 3 seconds (right).

The IP experiment was performed by ab307585 using HeLa cells. On the left the IP blot was probed with ab307585 and on the right the blot was probed by another anti-ADAR1 antibody (ab126745)(1:1000 dilution).