Recombinant Anti-Drosha antibody [EPR25336-93] (ab303544)

Blocking and dilution buffer and concentration: ntercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS. 

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

The identity of the bands between 80 kDa and 40 kDa are unknown.

False colour image of Western blot: Anti-Drosha antibody (ab303544) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red.
In Western blot, ab303544 was shown to bind specifically to Drosha. A band was observed at 180 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in Drosha knockout cell line ab266217 (knockout cell lysate ab257171). To generate this image, wild-type and Drosha knockout HEK-293T cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution. 

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

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

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

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

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labeling Drosha with AB303544 at 1/50 dilution (9.44 µg/mL), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (2 µg/mL) (Green). Confocal image showing mainly nuclear staining in NIH/3T3 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (2.5 µg/mL) (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 dilution (2 µg/mL).

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labeling Drosha with AB303544 at 1/50 dilution (1 µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled 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.

Drosha was immunoprecipitated from 0.35 mg Hela (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab303544 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab303544 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 μg (Inset)

Lane 2: ab303544 IP in Hela whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab303544 in Hela whole cell lysate

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

Exposure time: 180 seconds

Observed M_W (kDa): 180

Lysate was freshly made and used for IP immediately to minimize protein degradation.