Anti-cIAP2 antibody

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-cIAP2 antibody (AB23423)

ICC/IF image of ab23423 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab23423, 1μg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-cIAP2 antibody (AB23423)

ab23423 staining cIAP2 in HeLa cells treated with TMCB (ab120289), by ICC/IF. Decrease in cIAP2 expression correlates with increased concentration of TMCB, as described in literature.
The cells were incubated at 37°C for 10 minutes in media containing different concentrations of ab120289 (TMCB) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab23423 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

• FuncS

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Functional Studies - Anti-cIAP2 antibody (AB23423)

HeLa cells were incubated at 37°C for 24h with vehicle control (0 μM) and different concentrations of TMCB (ab120289). Decreased expression of cIAP2 in HeLa cells correlates with an increase in TMCB concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 40μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab23423 at 2 μg/ml and ab1801 at 2 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.

• WB

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Western blot - Anti-cIAP2 antibody (AB23423)

A doublet was observed in Daudi, Ramos and Raji whole cell lysates (lanes 1, 4 and 5). This data suggests that ab23423 might react with both cIAP1 and cIAP2 proteins. The predicted molecular weights of cIAP1 and cIAP2 are 69-kDa and 68-kDa respectively. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.

All lanes:

Western blot - Anti-cIAP2 antibody (ab23423) at 1 µg/mL

Lane 1:

Daudi (Human Burkitt's lymphoma cell line) Whole Cell Lysate at 10 µg/mL

Lane 2:

MOLT4 (Human acute lymphoblastic leukemia cell line) Whole Cell Lysate at 10 µg/mL

Lane 3:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg/mL

Lane 4:

Ramos (Human Burkitt's lymphoma cell line) Whole Cell Lysate at 10 µg/mL

Lane 5:

Raji (Human Burkitt's lymphoma cell line) Whole Cell Lysate at 10 µg/mL

Lane 6:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg/mL

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 68 kDa

Observed band size: 150 kDa,35 kDa,73 kDa,75 kDa

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Exposure time: 8min