All lanes : Anti-Bcl-XL antibody [E18] (ab32370) at 1/1000 dilution (purified)

Lane 1 : Jurkat cell lysate
Lane 2 : K562 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Observed band size: 26 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human endometrium tissue labelling Bcl-XL with purified ab32370 at 1/2000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit HRP (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 C6(Rat glial tumor glial cell) cells labeling Bcl-XL with purified ab32370 at 1/100 dilution (1.42 µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 dilution (2 µg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunocytochemistry/ Immunofluorescence analysis of NIH/3T3(Mouse embryonic fibroblast) cells labeling Bcl-XL with purified ab32370 at 1/100 dilution (1.42 µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 dilution (2 µg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

ab32370 (purified) at 1/20 dilution immunoprecipitating Bcl-XL in NIH/3T3 whole cell lysate.
Lane 1 (input): NIH/3T3(Mouse embryonic fibroblast) whole cell lysate 10µg
Lane 2 (+): ab32370 & NIH/3T3 whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32370 in NIH/3T3 whole cell lysate
For western blotting, ab32370 at 1/500 dilution (0.28 µg/ml)
and 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 NIH/3T3 (Mouse embryonic fibroblast) cells labelling Bcl-XL with ab32370 at 1/20 dilution (7.1 µg/ml) (Red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) (Black). Unlabeled control - Unlabelled cells (Blue).

Flow cytometry analysis of C6 (Rat glial tumor glial cell) cells labelling Bcl-XL with ab32370 at 1/20 dilution (7.1 µg/ml) (Red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) (Black). Unlabeled control - Unlabelled cells (Blue).

ICC/IF image of unpurified ab32370 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32370, 1/100) overnight at +4°C. The secondary antibody (green) was ab96899, goat anti-rabbit DyLight^® 488 (IgG; H+L) used at a 1/250 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

All lanes : Anti-Bcl-XL antibody [E18] (ab32370) at 1/1000 dilution (purified)

Lane 1 : C6 cell lysate
Lane 2 : RAW264.7 cell lysate
Lane 3 : PC-12 cell lysate
Lane 4 : NIH/3T3 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Observed band size: 26 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Flow cytometry analysis of Jurkat cells labelling Bcl-XL with purified ab32370 at 1/20 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

ab32370 (purified) at 1/30 immunoprecipitating Bcl-XL in Jurkat 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.

Anti-Bcl-XL antibody [E18] (ab32370) at 1/500 dilution (unpurified) + whole cell lysate prepared from a clinical sample of human breast cancer cells at 20 µg

Secondary
Goat anti-rabbit IgG-HRP at 1/1000 dilution

Developed using the ECL technique.

Observed band size: 26 kDa why is the actual band size different from the predicted?


Exposure time: 15 minutes


Patient recieved anthracycline and taxane neoadjuvant chemotherapy.

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Immunohistochemistry of human primary melanoma, staining Bcl-XL (red) with unpurified ab32370.
Antigen retrieval was performed in EDTA/Tris buffer (pH 8) before being blocked with 10%NGS for one hour at room temperature. Samples were incubated with primary antibody (1/50) at room temperature for one hour. An AlexaFluor®-conjugated anti-rabbit IgG was used as the secondary antibody.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human prostate carcinoma tissue labelling Bcl-XL with unpurified ab32370 at 1/50.

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Bcl-XL with purified ab32370 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, a goat anti-rabbit Alexa Fluor^® 488 (IgG; 1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

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

Overlay histogram showing DU145 cells stained with unpurified ab32370 (red line). The cells were fixed with 80% 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 (ab32370, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was a goat anti-rabbit DyLight® 488 (IgG; H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Equilibrium disassociation constant (K_D)
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