Anti-BRD2 antibody [BL-167-2A2]

• IP

Unknown

Immunoprecipitation - Anti-BRD2 antibody [BL-167-2A2] (AB243865)

BRD2 was immunoprecipitated from 0.5 mg HEK-293T whole cell lysate with ab243865 at 10 µl per reaction Western blot was performed on the immunoprecipitate using ab243865 at 1/1000 dilution. Lane 1 : ab243865 IP in HEK-293T cell lysate.

Lane 2 : Contol IgG in HEK-293T cell lysate.

Detection : Chemiluminescence with an exposure time of 3 minutes.

All lanes:

Immunoprecipitation - Anti-BRD2 antibody [BL-167-2A2] (ab243865)

Predicted band size: 88 kDa

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• WB

Lab

Western blot - Anti-BRD2 antibody [BL-167-2A2] (AB243865)

False colour image of Western blot : Anti-BRD2 antibody [BL-167-2A2] staining at 1/1000 dilution, shown in green; Mouse Anti-Nucleophosmin antibody [FC82291] (ab10530) loading control staining at 2 ug/mL imaged in ECL. In Western blot, ab243865 was shown to bind specifically to BRD2. A band was observed at 110 kDa in wild-type A549 cell lysates with no signal observed at this size in BRD2 knockout cell line. To generate this image, wild-type and BRD2 knockout A549 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 before development with Optiblot (ECL reagent ab133456) and imaged with 20 seconds exposure time. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and HRP conjugated Goat anti-Mouse (H+L) at 1/20000 dilution.

All lanes:

Western blot - Anti-BRD2 antibody [BL-167-2A2] (ab243865) at 1/1000 dilution

Lane 1:

Wild-type A549 cytoplasm cell lysate at 20 µg

Lane 2:

Wild-type A549 nuclear cell lysate at 20 µg

Lane 3:

BRD2 knockout A549 C7 cytoplasm cell lysate at 20 µg

Lane 4:

BRD2 knockout A549 C7 nuclear cell lysate at 20 µg

Lane 5:

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

Lane 6:

BRD2 knockout HEK-293T cell lysate at 20 µg

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• WB

Lab

Western blot - Anti-BRD2 antibody [BL-167-2A2] (AB243865)

Lanes 1-3 : Merged signal (red and green). Green - ab243865 observed at 110 kDa. Red - loading control ab8245 observed at 36 kDa.

ab243865 Anti-BRD2 antibody [BL-167-2A2] was shown to specifically react with BRD2 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267265 (knockout cell lysate ab257191) was used. Wild-type and BRD2 knockout samples were subjected to SDS-PAGE. ab243865 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours 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 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-BRD2 antibody [BL-167-2A2] (ab243865) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

BRD2 knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human BRD2 knockout HEK-293T cell line (<a href='/ko/products/cell-lines/human-brd2-knockout-hek-293t-cell-line-ab267265'>ab267265</a>)

Lane 3:

HeLa cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 88 kDa

Observed band size: 110 kDa

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• WB

Supplier Data

Western blot - Anti-BRD2 antibody [BL-167-2A2] (AB243865)

All lanes:

Western blot - Anti-BRD2 antibody [BL-167-2A2] (ab243865) at 1/1000 dilution

Lane 1:

HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 50 µg

Lane 2:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 50 µg

Lane 3:

Hep G2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 50 µg

Lane 4:

A549 (Human lung carcinoma cell line) whole cell lysate at 50 µg

Lane 5:

SW620 (Human colon carcinoma cell line) whole cell lysate at 50 µg

Lane 6:

SK-MEL-28 (Human melanoma cell line) whole cell lysate at 50 µg

Lane 7:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 50 µg

Secondary

All lanes:

Goat anti-rabbit IgG, HRP conjugated

Predicted band size: 88 kDa

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Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-BRD2 antibody [BL-167-2A2] (AB243865)

All lanes:

Western blot - Anti-BRD2 antibody [BL-167-2A2] (ab243865) at 1/1000 dilution

Lane 1:

RenCa (Mouse epithelial kidney cell line) whole cell lysate at 50 µg

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 50 µg

Lane 3:

C2C12 (Mouse myoblast cell line) whole cell lysate at 50 µg

Secondary

All lanes:

Goat anti-rabbit IgG, HRP conjugated

Predicted band size: 88 kDa

false

Exposure time: 3s