All lanes : Anti-Bcl-2 antibody [EPR17509] (ab182858) at 1/2000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : BCL2 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 26 kDa
Observed band size: 26 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab182858 observed at 26 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab182858 was shown to react with Bcl-2 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255364 (knockout cell lysate ab263752) was used. Wild-type HeLa and BCL2 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab182858 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling Bcl-2 with ab182858 at 1/1000 followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500.

Cytoplasm, nuclear membrane and nucleus staining on lymphocytes of Human tonsil tissue is observed.

Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody followed by ab97051 at 1/500.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow cytometric analysis of 4% paraformaldehyde-fixed Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling Bcl-2 with ab182858 at 1/250 (red) compared with a rabbit monoclonal IgG isotype control (ab172730) (black) and a unlabelled control (cells without incubation with primary antibody and secondary antibody (blue)). Goat anti rabbit IgG (FITC) at 1/500 was used as the secondary antibody.

All lanes : Anti-Bcl-2 antibody [EPR17509] (ab182858) at 1 µg/ml

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : BCL2 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : THP-1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 26 kDa
Observed band size: 26 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab182858 observed at 26 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab182858 was shown to specifically react with BCL2 when BCL2 knockout samples were used. Wild-type and BCL2 knockout samples were subjected to SDS-PAGE. Ab182858 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/10000 dilution respectively. Blots were developed 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Bcl-2 antibody [EPR17509] (ab182858) at 1/10000 dilution

Lane 1 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 20 µg
Lane 2 : WEHI-3 (mouse leukemia cell line) whole cell lysate at 20 µg
Lane 3 : Mouse hippocampus at 10 µg
Lane 4 : Mouse heart at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) at 1/2000 dilution

Predicted band size: 26 kDa
Observed band size: 26 kDa


Exposure time: 8 seconds

Blocking/Diluting buffer 5% NFDM/TBST

Immunohistochemical analysis of paraffin-embedded Human endometrial cancer tissue labeling Bcl-2 with ab182858 at 1/1000 followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500.

Cytoplasm, nuclear membrane and nucleus staining on lymphocytes and cancer cells of  Human endometrial cancer  tissue is observed.

Counter stained with Hematoxylin.
Negative control: Used PBS instead of primary antibody followed by ab97051 at 1/500.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-Bcl-2 antibody [EPR17509] (ab182858) at 1/20000 dilution

Lane 1 : Human tonsil lysate
Lane 2 : Human thymus lysate
Lane 3 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate
Lane 4 : U-937 (Human histiocytic lymphoma cells) whole cell lysate
Lane 5 : THP-1 (Human monocytic leukemia cells) whole cell lysate
Lane 6 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 7 : C2C12 (Mouse myoblast cell line) whole cell lysate
Lane 8 : WEHI-3 (Mouse leukemia cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

Developed using the ECL technique.

Predicted band size: 26 kDa
Observed band size: 26 kDa


Exposure time: 1 minute

Blocking and diluting buffer was 5% NFDM /TBST.

All lanes : Anti-Bcl-2 antibody [EPR17509] (ab182858) at 1/2000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse heart lysate
Lane 3 : Mouse kidney lysate
Lane 4 : Mouse spleen lysate
Lane 5 : NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

Developed using the ECL technique.

Predicted band size: 26 kDa
Observed band size: 26 kDa


Exposure time: 3 minutes

Blocking and diluting buffer was 5% NFDM /TBST.

All lanes : Anti-Bcl-2 antibody [EPR17509] (ab182858) at 1/2000 dilution

Lane 1 : Human fetal kidney lysate
Lane 2 : Human fetal spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Developed using the ECL technique.

Predicted band size: 26 kDa
Observed band size: 26 kDa


Exposure time: 3 minutes

Blocking and diluting buffer was 5% NFDM /TBST.

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling Bcl-2 with ab182858 at 1/1000 followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500.

Cytoplasm, nuclear membrane and nucleus staining on lymphocytes of Mouse spleen tissue is observed.

Counter stained with Hematoxylin.
Negative control: Used PBS instead of primary antibody followed by ab97051 at 1/500.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.