Anti-Brd4 antibody [EPR5150(2)] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Brd4 antibody [EPR5150(2)] - BSA and Azide free (AB182446)

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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Brd4 antibody [EPR5150(2)] - BSA and Azide free (AB182446)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab128874).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Brd4 antibody [EPR5150(2)] - BSA and Azide free (AB182446)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab128874).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Brd4 antibody [EPR5150(2)] - BSA and Azide free (AB182446)

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

• ICC/IF

AbReview32451****

Immunocytochemistry/ Immunofluorescence - Anti-Brd4 antibody [EPR5150(2)] - BSA and Azide free (AB182446)

Unpurified ab128874 (1/500 dilution) staining Brd4 in HeLa (Human epithelial cell line 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab128874).

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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Brd4 antibody [EPR5150(2)] - BSA and Azide free (AB182446)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab128874).

• WB

Lab

Western blot - Anti-Brd4 antibody [EPR5150(2)] - BSA and Azide free (AB182446)

This data was developed using ab128874, the same antibody clone in a different buffer formulation. 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)] (<a href='/en-us/products/primary-antibodies/brd4-antibody-epr51502-ab128874'>ab128874</a>)

Predicted band size: 152 kDa

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• WB

Lab

Western blot - Anti-Brd4 antibody [EPR5150(2)] - BSA and Azide free (AB182446)

This data was developed using the same antibody clone in a different buffer formulation (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)] (<a href='/en-us/products/primary-antibodies/brd4-antibody-epr51502-ab128874'>ab128874</a>) 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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• OI-RD Scanning

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OI-RD Scanning - Anti-Brd4 antibody [EPR5150(2)] - BSA and Azide free (AB182446)

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.