Anti-Drosha antibody [EPR25336-93]

• IP

Supplier Data

Immunoprecipitation - Anti-Drosha antibody [EPR25336-93] (AB303544)

Drosha was immunoprecipitated from 0.35 mg Hela (human cervical adenocarcinoma epithelial cell) whole cell lysate 10ug with ab303544 at 1/30 dilution (2ug in 0.35mg 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 10ug Lane 2 : ab303544 IP in Hela whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab303545 in Hela whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 180 seconds Lysate was freshly made and used for IP immediately to minimize protein degradation.

All lanes:

Immunoprecipitation - Anti-Drosha antibody [EPR25336-93] (ab303544) at 1/1000 dilution

All lanes:

Hela (human cervical adenocarcinoma epithelial cell) whole cell lysate

Secondary

All lanes:

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

Observed band size: 180 kDa

false

Exposure time: 180s

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Drosha antibody [EPR25336-93] (AB303544)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling Drosha with ab303544 at 1/50 (9.44 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 mainly nuclear staining in NIH/3T3 cell line. is observed. 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 Cyt

Supplier Data

Flow Cytometry - Anti-Drosha antibody [EPR25336-93] (AB303544)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling Drosha with ab303544 at 1/50 dilution (1ug) (Red) (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.

• WB

Lab

Western blot - Anti-Drosha antibody [EPR25336-93] (AB303544)

Western blot : Anti-DROSHA antibody [EPR25336-93] (ab303544) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab303544 was shown to bind specifically to DROSHA. A band was observed at 117-171 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in DROSHA knockout cell line. To generate this image, wild-type and DROSHA knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Drosha antibody [EPR25336-93] (ab303544) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human DROSHA knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-drosha-knockout-hct116-cell-line-ab287376'>ab287376</a>)

Lane 2:

DROSHA knockout HCT 116 cell lysate at 20 µg

Secondary

Lanes 1 - 2:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 2:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 117-171 kDa

false

• WB

Supplier Data

Western blot - Anti-Drosha antibody [EPR25336-93] (AB303544)

Blocking and dilution buffer and concentration : 5% NFDM/TBST.  Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes:

Western blot - Anti-Drosha antibody [EPR25336-93] (ab303544) at 1/1000 dilution

Lane 1:

A549 (human lung carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

C2C12 (mouse myoblast), whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 159 kDa

Observed band size: 180 kDa

false

Exposure time: 92s

• WB

Supplier Data

Western blot - 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.

All lanes:

Western blot - Anti-Drosha antibody [EPR25336-93] (ab303544) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lanes 1 - 2:

Western blot - Human DROSHA knockout HEK-293T cell lysate (<a href='/en-us/products/cell-lysates/human-drosha-knockout-hek-293t-cell-lysate-ab257171'>ab257171</a>) at 20 µg

Secondary

Lanes 1 - 2:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Lanes 1 - 2:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 159 kDa

Observed band size: 180 kDa

false

• WB

Supplier Data

Western blot - Anti-Drosha antibody [EPR25336-93] (AB303544)

Blocking and dilution buffer and concentration : 5% NFDM/TBST.  Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes:

Western blot - Anti-Drosha antibody [EPR25336-93] (ab303544) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 20 µg

Lane 2:

PC-12 (rat adrenal gland pheochromocytoma cell), whole cell lysate, at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 159 kDa

Observed band size: 180 kDa

false

Exposure time: 81s