Anti-Bcl-2 antibody [E17] (ab32124)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [E17] to Bcl-2
- Suitable for: WB, IHC-P, IP
- Knockout validated
- Reacts with: Human
Overview
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Product name
Anti-Bcl-2 antibody [E17]
See all Bcl-2 primary antibodies -
Description
Rabbit monoclonal [E17] to Bcl-2 -
Host species
Rabbit -
Specificity
Anti-Bcl-2 antibody [E17] (ab32124) recognises Bcl-2. It does not cross-react with other Bcl-2 family members. -
Tested Applications & Species
See all applications and species dataApplication Species IHC-P HumanIP HumanWB Human -
Immunogen
Synthetic peptide within Human Bcl-2 aa 50-150. The exact sequence is proprietary.
Database link: P10415 -
Positive control
- WB: MCF7, A431, Jurkat, HeLa, THP-1 and SH-SY5Y cell lysates; Wild type HAP1 whole cell lysate. IHC-P: Human B-cell lymphona and breast carcinoma tissues; Human UM xenografts; Human salivary glands; Human DLBCL U2932 cell line xenograft tissue. IP: Jurkat whole cell lysate (ab7899).
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General notes
Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
Abcam recommended secondaries - Goat Anti-Rabbit HRP (ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (ab150077).
See other anti-rabbit secondary antibodies that can be used with this antibody.
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
Dissociation constant (KD)
KD = 3.00 x 10 -11 M Learn more about KD -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
E17 -
Isotype
IgG -
Research areas
Images
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human B-cell lymphoma tissue labelling Bcl-2 with purified ab32124 at 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.
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All lanes : Anti-Bcl-2 antibody [E17] (ab32124) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : BCL2 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 26 kDa
Observed band size: 26 kDaLanes 1- 2: Merged signal (red and green). Green - ab32124 observed at 26 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab32124 was shown to react with BCL2 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255364 (knockout cell lysate ab263752) was used. Wild-type HeLa and BCL2 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32124 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
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Formaldehyde-fixed, paraffin-embedded human DLBCL U2932 cell line xenograft tissue stained for Bcl-2 using ab32124 at 1/200 dilution in immunohistochemical analysis, followed by Goat anti-Rabbit IgG Alexa Fluor® 555.
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All lanes : Anti-Bcl-2 antibody [E17] (ab32124)
Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : BCL2 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : THP-1 whole cell lysate
Lysates/proteins at 20 µg per lane.
Predicted band size: 26 kDaLanes 1 - 4: Merged signal (red and green). Green - ab32124 observed at 26 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab32124 was shown to specifically react with BCL2 when BCL2 knockout samples were used. Wild-type and BCL2 knockout samples were subjected to SDS-PAGE. Ab32124 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging. 3% milk used as blocking agent.
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ab32124 (purified) at 1/30 immunoprecipitating Bcl-2 in Jurkat (human T cell leukemia cell line from peripheral blood) 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.
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Bcl-2 expression determined by immunohistochemical analyses of the 4 human UM xenografts (between 3 to 5 tumors have been studied per condition).
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All lanes : Anti-Bcl-2 antibody [E17] (ab32124) at 1/1000 dilution (puriifed)
Lane 1 : MCF7 (human breast adenocarcinoma cell line) cell lysate
Lane 2 : A431 (human epidermoid carcinoma cell line) cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution
Predicted band size: 26 kDa
Observed band size: 26 kDaBlocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human B cell lymphoma tissue labelling Bcl-2 with unpurified ab32124. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.
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All lanes : Anti-Bcl-2 antibody [E17] (ab32124) at 1/200 dilution
Lane 1 : MCF7 (human breast adenocarcinoma cell line) cell lysate
Lane 2 : SK-BR-3 (human mammary gland adenocarcinoma cell line) cell lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/50000 dilution
Predicted band size: 26 kDa
Observed band size: 26 kDaBlocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.
MCF-7 cells express Bcl-2, while SK-BR-3 cells do not express Bcl-2 (PMID: 18430249)
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All lanes : Anti-Bcl-2 antibody [E17] (ab32124) at 1/10000 dilution (purified)
Lane 1 : Jurkat (human T cell leukemia cell line from peripheral blood) cell lysate
Lane 2 : HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 3 : SH-SY5Y (human neuroblastoma cell line from bone marrow) cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution
Predicted band size: 26 kDa
Observed band size: 26 kDaBlocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.
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Anti-Bcl-2 antibody [E17] (ab32124) at 1/1000 dilution (unpurified) + Jurkat (human T cell leukemia cell line from peripheral blood) cell lysate
Predicted band size: 26 kDa
Observed band size: 26 kDa
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Immunohistochemical analysis of Human salivary glands taken from patients with primary Sjögren's syndrome, staining Bcl-2 with unpurified ab32124.
Antigen retrieval was performed via heat mediation in a citrate buffer (pH 6). Sections were blocked using 2% BSA, 10% normal serum and permeabilized with 0.5% Triton X-100. Samples were incubated with primary antibody (1/100) for one hour at room temperature. An Alexa Fluor® 594-conjugated anti-rabbit IgG was used as the secondary antibody.
N.B. Panels B and D are higher magnifications of panels A and C, respectively. -
Equilibrium disassociation constant (KD)
Click here to learn more about KD -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling Bcl-2 with unpurified ab32124 at 1/200 dilution. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.
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