Anti-Smac/Diablo 抗体 [Y12] (ab32023)

Loading control: Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1/200000) (36KDa).

Blocking/Diluting buffer and concentration 5% NFDM/TBST

Low expression: spleen, brain, heart (PMID: 12058276)

Loading control: Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1/200000) (36KDa).

Blocking/Diluting buffer and concentration 5% NFDM/TBST

Immunohistochemistry analysis of paraffin-embedded mouse testis tissue sections labeling Smac/Diablo with ab32023 at 1/6000 dilution, incubated for 30 mins at room temperature followed by ab209101 (ready to use). Sections were counterstained with hematoxylin. Antigen retrieval was heat mediated with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Positive staining on mouse testis.

Immunohistochemistry analysis of paraffin-embedded mouse brain tissue sections labeling Smac/Diablo with ab32023 at 1/6000 dilution, incubated for 30 mins at room temperature followed by ab209101 (ready to use). Sections were counterstained with hematoxylin. Antigen retrieval was heat mediated with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Positive staining on mouse brain.

Immunocytochemistry/Immunofluorescence analysis of NIH/3T3 (mouse embryonic fibroblast) cells labelling Smac / Diablo with ab32023 at 1/100 dilution (green). Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100. ab150081, a Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (1/1000) was used as the secondary antibody. Anti-COX IV mouse monoclonal antibody - Mitochondrial Marker at 1/1000 was as the counterstain antibody followed by ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (magenta). Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Negative Control 1: ab32023 at 1/100 followed by ab150120 at 1/1000.
Negative Control 2: anti-COX IV antibody at 1/1000 followed by ab150081 at 1/1000.

Confocal image showing positive staining in mitochondria of NIH/3T3 cell line (shown in green).

Immunocytochemistry/Immunofluorescence analysis of C6 (rat glial tumor glial cell) cells labelling Smac / Diablo with ab32023 at 1/100 dilution (green). Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100. ab150081, a Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (1/1000) was used as the secondary antibody. Anti-COX IV mouse monoclonal antibody - Mitochondrial Marker at 1/1000 was as the counterstain antibody followed by ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (magenta). Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Negative Control 1: ab32023 at 1/100 followed by ab150120 at 1/1000.
Negative Control 2: anti-COX IV antibody at 1/1000 followed by ab150081 at 1/1000.

Confocal image showing positive staining in mitochondria of C6 cell line (shown in green).

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Smac / Diablo with ab32023 at 1/500. Cells were fixed with 100% methanol. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody.
Control: PBS only.
Nuclear counter stain: DAPI.

ab32023, at a 1/10 dilution, staining human Smac/Diablo in skin cancer tissue by immunohistochemistry, paraffin-embedded tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunofluorescence analysis of IMR32 cells, staining Smac / Diablo with ab32023. Cells were fixed with 4% paraformaldehyde before incubating with primary antibody. An Alexa Fluor^® conjugated anti-rabbit IgG was used as the secondary antibody.

Smac/Diablo was immunoprecipitated from 0.35 mg Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10 μg with ab32023 at 1/50 dilution (2μg). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution. 

Lane 1: Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10 μg

Lane 2: ab32023 IP in Jurkat whole cell lysate

Lane 3:Rabbit monoclonal IgG (ab172730) instead of ab32023 in Jurkat whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.