Anti-BRD2 抗体 [EPR7642] - ChIP Grade (ab139690)

All lanes : Anti-BRD2 antibody [EPR7642] - ChIP Grade (ab139690) at 1/1000 dilution

Lane 1 : Wild-type A549 ab288558 cytoplasm lysate
Lane 2 : Wild-type A549 ab288558 nuclear lysate
Lane 3 : BRD2 knockout A549 cytoplasm lysate
Lane 4 : BRD2 knockout A549 nuclear lysate
Lane 5 : Wild-type HEK-293T ab255553 cell lysate
Lane 6 : BRD2 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 88 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

False colour image of Western blot: Anti-BRD2 antibody [EPR7642] - ChIP Grade 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, ab139690 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 : Anti-BRD2 antibody [EPR7642] - ChIP Grade (ab139690) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : BRD2 knockout HEK293T cell lysate
Lane 3 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 88 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab139690 observed at 110 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab139690 Anti-BRD2 antibody [EPR7642]  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.  ab139690 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.

Chromatin was prepared from LNCaP cells according to the Abcam Dual X-ChIP protocol*. Cells were fixed with formaldehyde for 10 minutes.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab139690 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are located in the first kb of the transcribed region.
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: BRD2 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: A431 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab139690observed at 110 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab139690 was shown to specifically react with BRD2 when BRD2 knockout samples were used. Wild-type and BRD2 knockout samples were subjected to SDS-PAGE. ab139690 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 respectively and incubated overnight at 4°C. 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 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes : Anti-BRD2 antibody [EPR7642] - ChIP Grade (ab139690) at 1/1000 dilution

Lane 1 : MOLT4 cell lysate
Lane 2 : Jurkat cell lysate
Lane 3 : NCCIT cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Standard HRP labelled goat anti-rabbit at 1/2000 dilution

Developed using the ECL technique.

Predicted band size: 88 kDa

ab139690 staining Brd2 in wild-type HAP1 cells (top panel) and BRD2 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab139690 at 0.5μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Immunofluorescence analysis of HeLa cells labeling BRD2, with ab139690 at 1/250 dilution.

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling BRD2 with ab139690 at 1/250 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Overlay histogram showing HeLa cells stained with ab139690 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab139690, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.