Anti-Brd4 抗体 [EPR5150(2)]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling Brd4 with ab128874 at a concentration of 1 µg/ml. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins. ab128874 Anti-Brd4 antibody [EPR5150(2)] was incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin. Image inset shows absence of staining in secondary antibody only control.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Unpurified ab128874 at 1/100 dilution staining Brd4 in human colon carcinoma tissue by Immunohistochemistry.

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling Brd4 with ab128874 at a concentration of 3 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32 mins at 100°C with ULTRA cell conditioning solution (CC1, pH 8.5). ab128874 Anti-Brd4 antibody [EPR5150(2)] was incubated at 37°C for 16 mins. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

• WB

Lab

Western blot - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Western blot : Rabbit Monoclonal [EPR5150(2)] to Brd4 ab128874 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin (ab7291) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 200 kDa. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc 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 Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (whole lysate) at 20 µg

Lane 2:

Wild-type HAP1 (Nuclear fraction) at 20 µg

Lane 3:

Wild-type HAP1 (Membrane fraction) at 20 µg

Lane 4:

HEK-293T (Whole lysate) at 20 µg

Lane 5:

HeLa (Whole lysate) at 20 µg

Lane 6:

HeLa (Nuclear fraction) at 20 µg

Lane 7:

HeLa (Membrane fraction) at 20 µg

Secondary

Lanes 1 - 7:

Goat anti-Rabbit 800CW at 1/20000 dilution

Lanes 1 - 7:

Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 200 kDa

Observed band size: 200 kDa,50 kDa

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• WB

Supplier Data

Western blot - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Blocking and Diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/200 dilution

All lanes:

HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg/mL

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 152 kDa

Observed band size: 152 kDa

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• WB

Supplier Data

Western blot - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Blocking and Diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/1000 dilution

All lanes:

HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 152 kDa

Observed band size: 152 kDa

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• WB

Supplier Data

Western blot - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

All lanes:

Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/1000 dilution

All lanes:

NIH/3T3 (Mouse embryo fibroblast cell line) cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 152 kDa

Observed band size: 152 kDa

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• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Intracellular Flow Cytometry analysis of SW480 (Human colorectal adenocarcinoma epithelial cell) cells labeling Brd4 (red) with purified ab128874 at a 1/50 dilution (10μg/mL). Cells were fixed with 80% methanol and permeabilized with 0.1% Tween-20. A goat anti rabbit IgG (Alexa Fluorr^®488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (Black) (ab172730). Blue (unlabeled control) - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

AbReview32451****

Immunocytochemistry/ Immunofluorescence - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Unpurified ab128874 (1/500 dilution) staining Brd4 in HeLa cells from cervix adenocarcinoma) cells (green). Cells were fixed in paraformaldehyde permeabilized in 0.5% Triton X100/PBS and counterstained with DAPI in order to highlight the nucleus (red).

For further experimental details please refer to Abreview.

Image courtesy of an abreview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Immunocytochemistry/Immunofluorescence analysis of HepG2 (Human liver cell line) cells labeling Brd4 with purified ab128874 at 1/100 dilution (Panel A). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/200 dilution) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain(Panel B).

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Immunocytochemical analysis of HeLa cells showing co-localization of BRD4 using ab128874 at 1/500 dilution (red) with CDK9 (green) following infection with HSV-1. Cells were fixed with 4% paraformaldehyde (10 min at RT) and permeabilzed with 0.2% Triton X-100 (10 min). AlexaFluor^®-conjugated secondary antibodies were used at 1/1000 dilution. The nuclear counter stain is DAPI 9blue).

From Figure 5b of Ren et al.

Reproduced under the Creative Commons Licence fromRen et al PLoS Pathog. 2016 Oct; 12(10) : e1005950. Published online 2016 Oct 20. doi : 10.1371/journal.ppat.1005950 PMID : 27764245

Ren et al PLoS Pathog. 2016 Oct; 12(10): e1005950. Published online 2016 Oct 20. doi: 10.1371/journal.ppat.1005950

• WB

Lab

Western blot - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Different batches of ab128874 were tested on HeLa (Human cervix adenocarcinoma epithelial cell) lysate at 0.1 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 152 kDa.

All lanes:

Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874)

Predicted band size: 152 kDa

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• WB

Lab

Western blot - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Lanes 1 - 2 : Merged signal (red and green). Green - ab128874 observed at 220 kDa. Red - loading control Mouse anti Vinculin observed at 125 kDa.

ab128874 was shown to react with Brd4 in wild-type cells in Western blot with loss of signal observed in BRD4 knockout sample. Wild-type and BRD4 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab128874 and Mouse anti Vinculin overnight at 4 °C at a 1 in 200 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/200 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

BRD4 knockout HAP1 cell lysate at 20 µg

Predicted band size: 152 kDa

Observed band size: 220 kDa

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• WB

Lab

Western blot - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : Brd4 knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab128874 observed at 150 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab128874 was shown to recognize Brd4 when Brd4 knockout samples were used, along with additional cross-reactive bands. Wild-type and Brd4 knockout samples were subjected to SDS-PAGE. ab128874 and ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1/10 000 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 1/10000 dilution for 1 hour at room temperature before imaging.

Lane 1:

Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874)

Lanes 1 - 3:

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (<a href='/products/primary-antibodies/gapdh-antibody-6c5-loading-control-ab8245'>ab8245</a>) at 1/10000 dilution

Lane 1:

Wild-type HAP1 cell lysate (20 µg)

Lane 2:

Brd4 knockout HAP1 cell lysate (20 µg)

Lane 3:

HeLa cell lysate (20 µg)

Secondary

All lanes:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 152 kDa,36 kDa

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.