Human DROSHA knockout HEK-293T cell lysate

• WB

Unknown

Western blot - Human DROSHA knockout HEK-293T cell lysate (AB257171)

Lane 1 : Wild-type HEK293T cell lysate (20 μg)

Lane 2 : DROSHA knockout HEK293T cell lysate (20 μg)

Lane 3 : HeLa cell lysate (20 μg)

Lanes 1-3 : Merged signal (red and green). Green - ab183732 observed at 159 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab183732 Anti-Drosha antibody [EPR12794] was shown to specifically react with Drosha in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266217 (knockout cell lysate ab257171) was used. Wild-type and Drosha knockout samples were subjected to SDS-PAGE. ab183732 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Drosha antibody [EPR12794] (<a href='/en-us/products/primary-antibodies/drosha-antibody-epr12794-ab183732'>ab183732</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

DROSHA knockout HEK293T cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Predicted band size: 159 kDa

false

• Sanger seq

Unknown

Sanger Sequencing - Human DROSHA knockout HEK-293T cell lysate (AB257171)

Homozygous : 1 bp insertion in exon 4

• WB

Supplier Data

Western blot - Human DROSHA knockout HEK-293T cell lysate (AB257171)

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] (<a href='/en-us/products/primary-antibodies/drosha-antibody-epr25336-93-ab303544'>ab303544</a>) 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 (ab257171) 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 - Human DROSHA knockout HEK-293T cell lysate (AB257171)

All lanes:

Western blot - Anti-Drosha antibody [RM1063] (<a href='/en-us/products/primary-antibodies/drosha-antibody-rm1063-ab315100'>ab315100</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

Western blot - Human DROSHA knockout HEK-293T cell lysate (ab257171) at 20 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial 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 1/100000 dilution

Observed band size: 159 kDa,124 kDa

true

Exposure time: 125s

• WB

Supplier Data

Western blot - Human DROSHA knockout HEK-293T cell lysate (AB257171)

All lanes:

Western blot - Anti-Drosha antibody [RM1063] (<a href='/en-us/products/primary-antibodies/drosha-antibody-rm1063-ab315100'>ab315100</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

Western blot - Human DROSHA knockout HEK-293T cell lysate (ab257171) at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (800CW) and Goat Anti-Mouse IgG H&L (680RD) at 1/20000 dilution

Observed band size: 159 kDa,36 kDa

false