Anti-BRD3 antibody [2088C3a]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-BRD3 antibody [2088C3a] (AB50818)

Immunoflourescent staining of Hela cells using ab50818 Anti-BRD3 Antibody (1/10 dilution) as primary and AlexaFluor®488 Goat Anti-mouse IgG( 1/200 dilution )

• Flow Cyt

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Flow Cytometry - Anti-BRD3 antibody [2088C3a] (AB50818)

Flow Cytometric analysis using ab50818 (0.5ug). Secondary antibody used : AlexaFluor®488 Goat Anti-mouse IgG (0.25 µg)

• IP

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Immunoprecipitation - Anti-BRD3 antibody [2088C3a] (AB50818)

Immunoprecipitation of BRD3 from RIPA lysate of HeLa cells (500µg) using 5µg ab50818. Visualized by western blot using 1/50 ab50518 as primary and anti-mouse IgG secondary (1 : 2500). Predicted band size 80 KDa.

All lanes:

Immunoprecipitation - Anti-BRD3 antibody [2088C3a] (ab50818)

Predicted band size: 80 kDa

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• WB

Lab

Western blot - Anti-BRD3 antibody [2088C3a] (AB50818)

Western blot : Anti-BRD3 antibody [2088C3a] (ab50818) staining at 1/200 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab50818 was shown to bind specifically to BRD3. A band was observed at 90-100 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in BRD3 knockout cell line. To generate this image, wild-type and BRD3 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 HRP conjugated Goat anti-Mouse (H+L) and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-BRD3 antibody [2088C3a] (ab50818) at 1/200 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

BRD3 knockout HCT 116 cell lysate at 20 µg

Lane 3:

Wild-type HEK-293T ab255553 cell lysate at 20 µg

Lane 4:

BRD3 knockout HEK-293T ab260529 cell lysate at 20 µg

Secondary

Lanes 1 - 4:

HRP conjugated Goat anti-Mouse (H+L) at 1/20000 dilution

Lanes 1 - 4:

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

Observed band size: 90-100 kDa

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• WB

Lab

Western blot - Anti-BRD3 antibody [2088C3a] (AB50818)

Lanes 1-4 : Merged signal (red and green). Green - ab50818 observed at 100 kDa. Red - loading control ab181602 observed at 36 kDa.

ab50818 Anti-BRD3 antibody [2088C3a] was shown to specifically react with BRD3 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266793 (knockout cell lysate ab258335) was used. Wild-type and BRD3 knockout samples were subjected to SDS-PAGE. ab50818 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-BRD3 antibody [2088C3a] (ab50818) at 1/500 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

BRD3 knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human BRD3 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-brd3-knockout-hek-293t-cell-line-ab266793'>ab266793</a>)

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

HeLa cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Predicted band size: 80 kDa

Observed band size: 100 kDa

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• WB

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Western blot - Anti-BRD3 antibody [2088C3a] (AB50818)

All lanes:

Western blot - Anti-BRD3 antibody [2088C3a] (ab50818) at 1/50 dilution

All lanes:

HeLa whole cell lysate at 50 µg

Secondary

All lanes:

Anti-Mouse IgG antibody at 1/2500 dilution

Predicted band size: 80 kDa

Observed band size: 90 kDa

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• WB

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Western blot - Anti-BRD3 antibody [2088C3a] (AB50818)

All lanes:

Western blot - Anti-BRD3 antibody [2088C3a] (ab50818) at 1/50 dilution

All lanes:

NIH3T3 whole cell lysate at 50 µg

Secondary

All lanes:

Anti-Mouse IgG antibody at 1/2500 dilution

Predicted band size: 80 kDa

Observed band size: 90 kDa

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• WB

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Western blot - Anti-BRD3 antibody [2088C3a] (AB50818)

All lanes:

Western blot - Anti-BRD3 antibody [2088C3a] (ab50818) at 1/200 dilution

All lanes:

F2408 whole cell lysate at 50 µg

Secondary

All lanes:

Anti Mouse IgG antibody at 1/2500 dilution

Predicted band size: 80 kDa

Observed band size: 100 kDa

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