Anti-Bcl-XL antibody [E18]

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-XL antibody [E18] (AB32370)

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.

Image from Medic S & Ziman M PLoS One. 2010 Apr 22;5(4):e9977. Fig 5.; doi:10.1371/journal.pone.0009977; April 22 2010 PLoS ONE 5(4): e9977.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-XL antibody [E18] (AB32370)

The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Paraffin embedded human tonsil tissue slides were dewaxed, followed by heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, Epitope Retrieval ER2 Solution) for 20 mins. Endogenous peroxidase activity was blocked by Refine Detection Kit Peroxide Block for 10 mins. The sections were then incubated with ab32370 (1/1000) at room temperature for 30 mins, followed by a ready to use LeicaDS9800 (BOND Polymer Refine Detection Kit). The sections were counterstained with Hematoxylin. The images were taken with a Leica Aperio AT2.

ab32370 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in IHC-P. The image shows the staining intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Bcl-XL antibody [E18] (AB32370)

Intracellular 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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Bcl-XL antibody [E18] (AB32370)

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 22°C. The secondary antibody used was a goat anti-rabbit DyLight® 488 (IgG; H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Bcl-XL antibody [E18] (AB32370)

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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-XL antibody [E18] (AB32370)

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

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Bcl-XL antibody [E18] (AB32370)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-XL antibody [E18] (AB32370)

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.

• IP

Unknown

Immunoprecipitation - Anti-Bcl-XL antibody [E18] (AB32370)

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.

All lanes:

Immunoprecipitation - Anti-Bcl-XL antibody [E18] (ab32370)

Predicted band size: 26 kDa

Observed band size: 26 kDa

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• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Bcl-XL antibody [E18] (AB32370)

Flow cytometry analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labelling Bcl-XL with ab32370 at 1/20 dilution (7.1 © : g/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 Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Bcl-XL antibody [E18] (AB32370)

Flow cytometry analysis of C6 (Rat glial tumor glial cell) cells labelling Bcl-XL with ab32370 at 1/20 dilution (7.1 © : g/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).

• IP

Unknown

Immunoprecipitation - Anti-Bcl-XL antibody [E18] (AB32370)

ab32370 (purified) at 1/500 dilution (0.28 © : g/ml) immunoprecipitating Bcl-XL in NIH/3T3 whole cell lysate.
Lane 1 (input) : NIH/3T3(Mouse embryonic fibroblast) whole cell lysate 10© : g
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, VeriBlot for IP secondary antibody (HRP) (ab131366) was used as the secondary antibody at 1/1000 dilution.
Blocking and diluting buffer : 5% NFDM /TBST .

All lanes:

Immunoprecipitation - Anti-Bcl-XL antibody [E18] (ab32370)

Predicted band size: 26 kDa

false

• WB

Lab

Western blot - Anti-Bcl-XL antibody [E18] (AB32370)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of K-562 whole cell lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab32370 (1/1000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab32370 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

All lanes:

Western blot - Anti-Bcl-XL antibody [E18] (ab32370) at 1/1000 dilution

All lanes:

K562 whole cell lysate at 15 µg with NDFM/TBST

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

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Exposure time: 80s

• WB

Lab

Western blot - Anti-Bcl-XL antibody [E18] (AB32370)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Bcl-XL antibody [E18] (ab32370) at 1/1000 dilution

Lane 1:

Jurkat cell lysate at 20 µg

Lane 2:

K562 cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 26 kDa

Observed band size: 26 kDa

false

• WB

AbReview24938****

Western blot - Anti-Bcl-XL antibody [E18] (AB32370)

Patient recieved anthracycline and taxane neoadjuvant chemotherapy.

All lanes:

Western blot - Anti-Bcl-XL antibody [E18] (ab32370) at 1/500 dilution

All lanes:

whole cell lysate prepared from a clinical sample of human breast cancer cells at 20 µg

Secondary

All lanes:

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

Predicted band size: 26 kDa

Observed band size: 26 kDa

true

Exposure time: 15min

Image courtesy of an anonymous Abreview.

• WB

Lab

Western blot - Anti-Bcl-XL antibody [E18] (AB32370)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Bcl-XL antibody [E18] (ab32370) at 1/1000 dilution

Lane 1:

C6 cell lysate at 20 µg

Lane 2:

RAW264.7 cell lysate at 20 µg

Lane 3:

PC-12 cell lysate at 20 µg

Lane 4:

NIH/3T3 cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 26 kDa

Observed band size: 26 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Bcl-XL antibody [E18] (AB32370)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.