Anti-Survivin antibody [EP2880Y] (ab76424) at 1/5000 dilution (purified) + A549 (Human lung carcinoma epithelial cell) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 16 kDa



Blocking and diluting buffer : 5% NFDM/TBST

ab76424 (purified) at 1:150 dilution (5µg) immunoprecipitating Survivin in Ramos whole cell lysate.
Lane 1 (input): Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysate 10µg
Lane 2 (+): ab76424 & Ramos whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab76424 in Ramos whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Survivin with purified ab76424 at 1:500 dilution (5 µg/ml) (red). Cells were fixed with 80% methanol. A Goat anti rabbit IgG (Alexa Fluor® 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).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human bladder carcinoma tissue sections labeling Survivin with Purified ab76424 at 1:1000 dilution (2.52 µg/ml). Heat mediated antigen retrieval was performed using Tris/EDTA Buffer, pH 9.0. Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP)
secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.

Immunofluorescent staining of MCF-7 cells labelling Bcl-2 with purified ab76424 at 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin (1/1000) using ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) 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).

All lanes : Anti-Survivin antibody [EP2880Y] (ab76424) at 1/5000 dilution (purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 16 kDa



Blocking and diluting buffer : 5% NFDM/TBST

All lanes : Anti-Survivin antibody [EP2880Y] (ab76424) at 1/10000 dilution (unpurified)

Lane 1 : HeLa lysates
Lane 2 : Jurkat lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : goat anti-rabbit HRP at 1/1000 dilution

Predicted band size: 16 kDa
Observed band size: 16 kDa

Unpurified ab76424 showing positive staining in Fetal kidney tissue. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Overlay histogram showing HeLa cells stained with unpurified ab76424 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab76424, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in HeLa cells fixed with 4% paraformaldehyde/permeabilized with 0.1% PBS-Tween 20 used under the same conditions.

Unpurified ab76424 at 1/250 dilution staining Survivin in human colonic adenocarcinoma by Immunohistochemistry, Paraffin-embedded tissue. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Unpurified ab76424 at 1/250 dilution staining Survivin in human urinary bladder carcinoma by Immunohistochemistry, Paraffin-embedded tissue. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Unpurified ab76424 showing positive staining in breast carcinoma tissue. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Unpurified ab76424 showing positive staining in cervical carcinoma tissue. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Unpurified ab76424 showing positive staining in fetal liver tissue. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Unpurified ab76424 showing positive staining in normal tonsil tissue. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Immunocytochemistry/Immunofluorescence analysis of MCF-7 cells labelling Survivin with unpurified ab76424 at 1/100. Cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. Cells were incubated with the primary antibody overnight. An Alexa Fluor^® 555-conjugated anti-rabbit IgG was used as the secondary antibody. Left - Survivin, Right - DAPI.

Standard curve for Survivin (Analyte: ab87202); dilution range 1pg/ml to 1µg/ml using Capture Antibody ab27468 at 1µg/ml and Detector Antibody abRabbit monoclonal [EP2880Y] to Survivin (ab76424) at 0.5µg/ml. Concentration of Rabbit monoclonal [EP2880Y] to Survivin (ab76424) may vary from lot to lot; please use this curve as guideline.

Equilibrium disassociation constant (K_D)
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ab76424 staining Survivin in the Hela cell line from Human cervix by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.5% Triton X-100. Samples were incubated with primary antibody (1/500 in PBS) for 1 hour at 22°C. Ab150081 (1/200) was used as the secondary antibody.

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