Anti-BDNF 抗体 [EPR1292]

• WB

Lab

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

Predicted band size: 27 kDa

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

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BDNF antibody [EPR1292] (AB108319)

Immunohistochemical analysis of formalin fixed paraffin embedded human brain (cerebrum) labelling BDNF with ab108319 at a concentration of 0.56 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). ab108319 Anti-BDNF antibody [EPR1292] 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.

• Flow Cyt (Intra)

Lab

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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BDNF antibody [EPR1292] (AB108319)

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.

• IHC-P

Lab

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)

• ICC/IF

AbReview56343****

Immunocytochemistry/ Immunofluorescence - Anti-BDNF antibody [EPR1292] (AB108319)

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.

This image is courtesy of an Abreview submitted by Kirk McManus (1894904)

• IHC-Fr

Lab

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^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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-BDNF antibody [EPR1292] (AB108319)

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.

• IHC-Fr

Unknown

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.

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-BDNF antibody [EPR1292] (AB108319)

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.

• WB

Lab

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

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• WB

Lab

Western blot - Anti-BDNF antibody [EPR1292] (AB108319)

Different batches of ab108319 were tested on Mouse brain lysate at 0.3 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 14-45 kDa.

All lanes:

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

Predicted band size: 27 kDa

false

• WB

Lab

Western blot - Anti-BDNF antibody [EPR1292] (AB108319)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of mouse brain lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab108319 (1/1000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab108319 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

All lanes:

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

All lanes:

Mouse brain lysate at 15 µg with NDFM/TBST

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

false

Exposure time: 5s

• WB

Lab

Western blot - Anti-BDNF antibody [EPR1292] (AB108319)

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

ab213204 was used as a His tag loading control.

All lanes:

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

Lane 1:

293T cells transfected with an empty vector containing a His tag whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a Human BDNF expression vector containing a His-tag whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a Human NGF expression vector containing a His-tag whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a Human NTF3 expression vector containing a His-tag whole cell lysate at 20 µg

Lane 5:

293T cells transfected with a Human NTF4 expression vector containing a His-tag whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 35 kDa

false

Exposure time: 1s