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

Lane 1 : Human hippocampus lysate
Lane 2 : Rat hippocampus lysate
Lane 3 : Mouse hippocampus lysate

Lysates/proteins at 20 µg per lane.

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

Performed under reducing conditions.

Predicted band size: 15 kDa
Additional bands at: 15 kDa (possible mature (processed) protein), 28 kDa (possible multimer), 35 kDa, 45 kDa (possible immature (unprocessed)). We are unsure as to the identity of these extra bands.

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 : Anti-BDNF antibody [EPR1292] (ab108319) at 1/1000 dilution (Purified)

Lane 1 : Human brain lysates with 5% NFDM/TBST
Lane 2 : Mouse brain lysates with 5% NFDM/TBST
Lane 3 : Rat brain lysates with 5% NFDM/TBST
Lane 4 : Human hippocampus lysates with 5% NFDM/TBST
Lane 5 : Mouse hippocampus lysates with 5% NFDM/TBST
Lane 6 : Rat hippocampus lysates with 5% NFDM/TBST
Lane 7 : Human cerebellum lysates with 5% NFDM/TBST
Lane 8 : Mouse cerebellum lysates with 5% NFDM/TBST
Lane 9 : Rat cerebellum lysates with 5% NFDM/TBST

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 15 kDa
Observed band size: 15-45 kDa why is the actual band size different from the predicted?

IHC image of BDNF staining in a section of frozen normal human cerebral cortex performed on a Leica BOND^TM 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) 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) 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 analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling BDNF with purified ab108319 at 1:30 dilution (10 µg/ml) (red). Cells were fixed with 80% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 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).

Immunohistochemistry (Frozen sections) analysis of mouse cerebrum 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.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling BDNF with Purified ab108319 at 1:500 (0.6 µg/ml). Cells were fixed in 100% Methanol and permeabilized with None. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemical analysis of paraffin-embedded human brain tissue using unpurified ab108319 at 1/100 dilution.

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

Immunocytochemistry/ Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) cells labelling BDNF with unpurified ab108319 at a dilution of 1/750. Cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton-X100 in PBS. ab150081 (1/200) was used as the secondary antibody.

The antibody produces a strong, golgi-associated labelling pattern in both PF and MeOH fixed samples.

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