Recombinant Anti-Bcl-2 antibody [E17] (ab32124)

Lane 1: Wild type HAP1 whole cell lysate (20 µg)              
Lane 2: BCL2 knockout  HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: THP-1 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab32124 observed at 26 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32124 was shown to specifically react with BCL2 when BCL2 knockout samples were used. Wild-type and BCL2 knockout samples were subjected to SDS-PAGE.  Ab32124 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1000 dilution 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. 3% milk used as blocking agent.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human B-cell lymphoma tissue labelling Bcl-2 with purified ab32124 at 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunocytochemistry/Immunofluorescence analysis of MCF7 (human breast adenocarcinoma cell line) cells labelling Bcl-2 with unpurified ab32124 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin antibody (1/500) using ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) as the secondary. Nuclei counterstained with DAPI (blue).

Control: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Formaldehyde-fixed, paraffin-embedded human DLBCL U2932 cell line xenograft tissue stained for Bcl-2 using ab32124 at 1/200 dilution in immunohistochemical analysis, followed by Goat anti-Rabbit IgG Alexa Fluor^® 555.

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Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Bcl-2 expression determined by immunohistochemical analyses of the 4 human UM xenografts (between 3 to 5 tumors have been studied per condition).

ab32124 (purified) at 1/30 immunoprecipitating Bcl-2 in Jurkat (human T cell leukemia cell line from peripheral blood) cell lysate (Lane 1). Lane 2 - PBS. For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human B cell lymphoma tissue labelling Bcl-2 with unpurified ab32124.

Methanol-fixed HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained for Bcl-2 using ab32124 at 1/200 dilution in ICC/IF, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor­ 488) preadsorbed (ab150081). Counter-stained with DAPI.

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Immunocytochemistry/Immunofluorescence analysis of MCF7 (human breast adenocarcinoma cell line) cells labelling Bcl-2 with purified ab32124 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin (1/500) using ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) as the secondary antibody. Nuclei counterstained with DAPI (blue). 

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

Equilibrium disassociation constant (K_D)

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Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

MCF-7 cells express Bcl-2, while SK-BR-3 cells do not express Bcl-2 (PMID: 18430249)

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Immunohistochemical analysis of Human salivary glands taken from patients with primary Sjögren's syndrome, staining Bcl-2 with unpurified ab32124.
Antigen retrieval was performed via heat mediation in a citrate buffer (pH 6). Sections were blocked using 2% BSA, 10% normal serum and permeabilized with 0.5% Triton X-100. Samples were incubated with primary antibody (1/100) for one hour at room temperature. An Alexa Fluor^® 594-conjugated anti-rabbit IgG was used as the secondary antibody.
N.B. Panels B and D are higher magnifications of panels A and C, respectively.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling Bcl-2 with unpurified ab32124 at 1/200 dilution.