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

Performed under reducing conditions.

Predicted band size: 152 kDa

Observed band size: 220 kDa

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 (ab8245) 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 (ab216776) at 1/10000 dilution

Predicted band size: 152 kDa, 36 kDa

This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab128874 overnight at 4°C. Antibody binding was detected using the Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773. Loading control to GAPDH ab8245 antibody binding was detected using the Goat anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

Merged signal (red and green). Green Anti-Brd4 antibody [EPR5150(2)] ab128874 observed at 150 kDa using Goat anti-Rabbit IgG H&L (IRDye® 800CW)-ab216773 as secondary antibody. Red - Anti-GAPDH antibody loading control, ab8245, observed at 37 kDa,

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 from Ren 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

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.

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).

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 (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) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain(Panel B).

Western blot - Anti-BDNF antibody [EPR1292] (ab108319)

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with unpurified ab108319 (1/1000) overnight at 4°C. ab8245 (mouse anti-GAPDH; 0.05 ug/mL) was included as a loading control. Antibody binding was detected using goat anti-rabbit IgG IR-680 (green) and goat anti-mouse IgG IR800 (red) at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx

All lanes: Western blot - Anti-BDNF antibody [EPR1292] (ab108319) at 1/1000 dilution

Lane 1: Human hippocampus lysate at 20 µg

Lane 2: Rat hippocampus lysate at 20 µg

Lane 3: Mouse hippocampus lysate at 20 µg

Secondary

All lanes: Gt anti Rb IR680 at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 27 kDa

Observed band size: 15 kDa, 28 kDa, 35 kDa, 45 kDa

Western blot - Anti-BDNF antibody [EPR1292] (ab108319)

All lanes: Western blot - Anti-BDNF antibody [EPR1292] (ab108319) at 1/1000 dilution

Lane 1: Human brain lysates at 20 µg

Lane 2: Mouse brain lysates at 20 µg

Lane 3: Rat brain lysates at 20 µg

Lane 4: Human hippocampus lysates at 20 µg

Lane 5: Mouse hippocampus lysates at 20 µg

Lane 6: Rat hippocampus lysates at 20 µg

Lane 7: Human cerebellum lysates at 20 µg

Lane 8: Mouse cerebellum lysates at 20 µg

Lane 9: Rat cerebellum lysates at 20 µg

Secondary

All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 27 kDa

Observed band size: 15-45 kDa

Immunohistochemistry (Frozen sections) - Anti-BDNF antibody [EPR1292] (ab108319)

IHC image of BDNF staining in a section of frozen normal human cerebral cortex performed on a Leica BOND™ system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab108319, 1/200 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Immunohistochemistry (Frozen sections) - Anti-BDNF antibody [EPR1292] (ab108319)

Immunohistochemistry (Frozen sections) analysis of rat cerebral cortex tissue sections labeling BDNF with Purified ab108319 at 1/100 (2.8 µg/ml).Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. DAPI was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BDNF antibody [EPR1292] (ab108319)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human bladder cancer tissue sections labeling BDNF with Purified ab108319 at 1:500 dilution (0.56 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Flow Cytometry (Intracellular) - Anti-BDNF antibody [EPR1292] (ab108319)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling BDNF with purified ab108319 at 1/30 dilution (10μg/ml) (red). Cells were fixed with 80% methanol. A Goat anti rabbit IgG (Alexa Fluorr® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).