Immunoprecipitation - Anti-Vinculin antibody [EPR8185] (ab129002)

ab129002 (purified) at 1/20 immunoprecipitating vinculin in HeLa cells. Lane 1: HeLa whole cell lysate (10 μg). Lane 2: HeLa whole cell lysate (10 μg). Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab129002 in HeLa whole cell lysate. For western blotting, a HRP-conjugated goat anti-rabbit antibody was used as the secondary antibody (1/1000).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

All lanes: Immunoprecipitation - Anti-Vinculin antibody [EPR8185] (ab129002)

Predicted band size: 124 kDa

Immunocytochemistry/ Immunofluorescence - Anti-Vinculin antibody [EPR8185] (ab129002)

Immunofluorescent staining of HEK293 cells (fixed in 4% PFA, permeabilized with 0.1% Triton X 100) using purified ab129002 at a dilution of 1/50. An Alexa Fluor® 488 goat anti-rabbit antibody (ab150077) was used as the secondary at a dilution of 1/1000 and the cells were counter stained with DAPI. The negative controls are shown in the bottom middle and right hand panels. For negative control 1, the primary was used and then goat anti-mouse IgG was used at a dilution of 1/500. For negative control 2, a mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077) were used.

Western blot - Anti-Vinculin antibody [EPR8185] (ab129002)

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

All lanes: Western blot - Anti-Vinculin antibody [EPR8185] (ab129002) at 1/10000 dilution

Lane 1: HepG2 cell lysate at 20 µg

Lane 2: HeLa cell lysate at 20 µg

Lane 3: PC3 cell lysate at 20 µg

Secondary

All lanes: HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 124 kDa

Observed band size: 124 kDa

Flow Cytometry (Intracellular) - Anti-Vinculin antibody [EPR8185] (ab129002)

Intracellular Flow Cytometry analysis of 293 (human embryonic kidney epithelial) cells labeling Vinculin (red) with ab129002 at a 1/200 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluorr® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.

Western blot - Anti-Vinculin antibody [EPR8185] (ab129002)

Different batches of ab129002 were tested on HepG2 (Human hepatocellular carcinoma epithelial cell) lysate at 1.0 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 124 kDa.

All lanes: Western blot - Anti-Vinculin antibody [EPR8185] (ab129002)

Predicted band size: 124 kDa

Western blot - Anti-Vinculin antibody [EPR8185] (ab129002)

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

All lanes: Western blot - Anti-Vinculin antibody [EPR8185] (ab129002) at 1/20000 dilution

Lane 1: C6 cell lysate at 10 µg

Lane 2: rat kidney cell lysate at 10 µg

Secondary

All lanes: HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 124 kDa

Observed band size: 124 kDa

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).