Anti-BRD2 antibody [EPR7642] - BSA and Azide free

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRD2 antibody [EPR7642] - BSA and Azide free (AB222393)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab139690).

Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-BRD2 antibody [EPR7642] - BSA and Azide free (AB222393)

Immunofluorescence analysis of HeLa cells labeling BRD2, with ab139690 at 1/250 dilution.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab139690).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-BRD2 antibody [EPR7642] - BSA and Azide free (AB222393)

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. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab139690).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-BRD2 antibody [EPR7642] - BSA and Azide free (AB222393)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab139690).

• ChIP

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ChIP - Anti-BRD2 antibody [EPR7642] - BSA and Azide free (AB222393)

Chromatin was prepared from HT-29 cells according to the Abcam 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
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab139690).

• IP

Lab

Immunoprecipitation - Anti-BRD2 antibody [EPR7642] - BSA and Azide free (AB222393)

This data was developed using ab139690, the same antibody clone in a different buffer formulation.

BRD2 was immunoprecipitated from HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate 10 μg with ab139690 1 : 1000 dilution (0.558 μg/ml). Western blot was performed on the immunoprecipitate using ab139690 at 1/1000 dilution. Capture antibody, 1 : 30 dilution (2μg in 0.35mg lysates).

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Lysate was freshly made and used for Immunoprecipitation immediately to minimize protein degradation.

All lanes:

Immunoprecipitation - Anti-BRD2 antibody [EPR7642] - ChIP Grade (<a href='/en-us/products/primary-antibodies/brd2-antibody-epr7642-chip-grade-ab139690'>ab139690</a>) at 1/1000 dilution

Lanes 1 - 2:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/brd2-antibody-epr7642-chip-grade-ab139690'>ab139690</a> in HeLa whole cell lysate at 10 µg

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: 110 kDa

false

Exposure time: 58s

• WB

Lab

Western blot - Anti-BRD2 antibody [EPR7642] - BSA and Azide free (AB222393)

This data was developed using the same antibody clone in a different buffer formulation (ab139690).

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.

All lanes:

Western blot - Anti-BRD2 antibody [EPR7642] - ChIP Grade (<a href='/en-us/products/primary-antibodies/brd2-antibody-epr7642-chip-grade-ab139690'>ab139690</a>) 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='/en-us/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='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 126 kDa,19 kDa,20 kDa,26 kDa,32 kDa,34 kDa,44 kDa,88 kDa

Observed band size: 110 kDa,26 kDa,50 kDa

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