Validated using a knockout cell line
Recombinant
RabMAb

Anti-Bcl-2 antibody [EPR17509] (ab182858)

Overview

  • Product name
    Anti-Bcl-2 antibody [EPR17509]
    See all Bcl-2 primary antibodies
  • Description
    Rabbit monoclonal [EPR17509] to Bcl-2
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, ICC/IF, Flow Cyt, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Recombinant fragment within Human Bcl-2 aa 1 to the C-terminus. The exact sequence is proprietary.
    Database link: P10415

  • Positive control
    • WB: Human tonsil lysate, Human thymus lysate; Jurkat, U-937, THP-1, HeLa, C2C12, WEHI -3 and NIH/3T3 whole cell lysates; Mouse brain lysate, Mouse heart lysate, Mouse kidney lysate, Mouse spleen lysate, Human fetal kidney lysate, Human fetal spleen lysate. IHC-P: Human tonsil tissue, Human endometrial cancer tissue, Mouse spleen tissue. ICC/IF: Jurkat cells. Flow Cyt: Jurkat cells.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    This product is a recombinant rabbit monoclonal antibody.

Applications

Our Abpromise guarantee covers the use of ab182858 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/2000. Detects a band of approximately 26 kDa (predicted molecular weight: 26 kDa).
ICC/IF 1/150.
Flow Cyt 1/250.
IHC-P 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Target

  • Function
    Suppresses apoptosis in a variety of cell systems including factor-dependent lymphohematopoietic and neural cells. Regulates cell death by controlling the mitochondrial membrane permeability. Appears to function in a feedback loop system with caspases. Inhibits caspase activity either by preventing the release of cytochrome c from the mitochondria and/or by binding to the apoptosis-activating factor (APAF-1). May attenuate inflammation by impairing NLRP1-inflammasome activation, hence CASP1 activation and IL1B release (PubMed:17418785).
  • Tissue specificity
    Expressed in a variety of tissues.
  • Involvement in disease
    A chromosomal aberration involving BCL2 has been found in chronic lymphatic leukemia. Translocation t(14;18)(q32;q21) with immunoglobulin gene regions. BCL2 mutations found in non-Hodgkin lymphomas carrying the chromosomal translocation could be attributed to the Ig somatic hypermutation mechanism resulting in nucleotide transitions.
  • Sequence similarities
    Belongs to the Bcl-2 family.
  • Domain
    BH1 and BH2 domains are required for the interaction with BAX and for anti-apoptotic activity.
    The BH4 motif is required for anti-apoptotic activity and for interaction with RAF1 and EGLN3.
    The loop between motifs BH4 and BH3 is required for the interaction with NLRP1.
  • Post-translational
    modifications
    Phosphorylation/dephosphorylation on Ser-70 regulates anti-apoptotic activity. Growth factor-stimulated phosphorylation on Ser-70 by PKC is required for the anti-apoptosis activity and occurs during the G2/M phase of the cell cycle. In the absence of growth factors, BCL2 appears to be phosphorylated by other protein kinases such as ERKs and stress-activated kinases. Phosphorylated by MAPK8/JNK1 at Thr-69, Ser-70 and Ser-87, wich stimulates starvation-induced autophagy. Dephosphorylated by protein phosphatase 2A (PP2A).
    Proteolytically cleaved by caspases during apoptosis. The cleaved protein, lacking the BH4 motif, has pro-apoptotic activity, causes the release of cytochrome c into the cytosol promoting further caspase activity.
    Monoubiquitinated by PARK2, leading to increase its stability. Ubiquitinated by SCF(FBXO10), leading to its degradation by the proteasome.
  • Cellular localization
    Mitochondrion outer membrane. Nucleus membrane. Endoplasmic reticulum membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • Apoptosis regulator Bcl 2 antibody
    • Apoptosis regulator Bcl-2 antibody
    • Apoptosis regulator Bcl2 antibody
    • AW986256 antibody
    • B cell CLL/lymphoma 2 antibody
    • B cell leukemia/lymphoma 2 antibody
    • Bcl-2 antibody
    • Bcl2 antibody
    • BCL2_HUMAN antibody
    • C430015F12Rik antibody
    • D630044D05Rik antibody
    • D830018M01Rik antibody
    • Leukemia/lymphoma, B-cell, 2 antibody
    • Oncogene B-cell leukemia 2 antibody
    • PPP1R50 antibody
    • Protein phosphatase 1, regulatory subunit 50 antibody
    see all

Images

  • Lane 1: Wild type HAP1 whole cell lysate (20 µg)
    Lane 2: BCL2 knockout HAP1 whole cell lysate (20 µg)
    Lane 3: HeLa whole cell lysate (20 µg)
    Lane 4: THP-1 whole cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab182858 observed at 26 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab182858 was shown to specifically react with BCL2 when BCL2 knockout samples were used. Wild-type and BCL2 knockout samples were subjected to SDS-PAGE. Ab182858 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml 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.

  • Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling Bcl-2 with ab182858 at 1/1000 followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500.

    Cytoplasm, nuclear membrane and nucleus staining on lymphocytes of Human tonsil tissue is observed.

    Counter stained with Hematoxylin.

    Negative control: Used PBS instead of primary antibody followed by ab97051 at 1/500.

  • Flow cytometric analysis of 4% paraformaldehyde-fixed Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling Bcl-2 with ab182858 at 1/250 (red) compared with a rabbit monoclonal IgG isotype control (ab172730) (black) and a unlabelled control (cells without incubation with primary antibody and secondary antibody (blue)). Goat anti rabbit IgG (FITC) at 1/500 was used as the secondary antibody.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling Bcl-2 with ab182858 at 1/150, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 (green).

    Confocal image showing cytoplasmic and weak nuclear staining on Jurkat cells line.

    The nuclear counterstain is DAPI (blue).

    Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 (red).

    The negative controls are as follows:-
    -ve control 1 - ab182858 at 1/150 followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000.
    -ve control 2. - ab7291 (anti-Tubulin mouse mAb) at 1/1000 followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000.

  • All lanes : Anti-Bcl-2 antibody [EPR17509] (ab182858) at 1/10000 dilution

    Lane 1 : NIH/3T3 (Mouse fibroblast) whole cell lysate at 20 µg
    Lane 2 : WEHI-3 (Mouse myelomonocyte from leukemia) whole cell lysate at 20 µg
    Lane 3 : Mouse hippocampus at 10 µg
    Lane 4 : Mouse heart at 10 µg

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) at 1/2000 dilution

    Predicted band size: 26 kDa
    Observed band size: 26 kDa


    Exposure time: 8 seconds


    Blocking/Diluting buffer 5% NFDM/TBST

  • Immunohistochemical analysis of paraffin-embedded Human endometrial cancer tissue labeling Bcl-2 with ab182858 at 1/1000 followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500.

    Cytoplasm, nuclear membrane and nucleus staining on lymphocytes and cancer cells of  Human endometrial cancer  tissue is observed.

    Counter stained with Hematoxylin.
    Negative control: Used PBS instead of primary antibody followed by ab97051 at 1/500.

  • All lanes : Anti-Bcl-2 antibody [EPR17509] (ab182858) at 1/20000 dilution

    Lane 1 : Human tonsil lysate
    Lane 2 : Human thymus lysate
    Lane 3 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate
    Lane 4 : U-937 (Human histiocytic lymphoma cells) whole cell lysate
    Lane 5 : THP-1 (Human monocytic leukemia cells) whole cell lysate
    Lane 6 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
    Lane 7 : C2C12 (Mouse myoblast cell line) whole cell lysate
    Lane 8 : WEHI-3 (Mouse leukemia cell line) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

    Developed using the ECL technique.

    Predicted band size: 26 kDa
    Observed band size: 26 kDa


    Exposure time: 1 minute


    Blocking and diluting buffer was 5% NFDM /TBST.

  • All lanes : Anti-Bcl-2 antibody [EPR17509] (ab182858) at 1/2000 dilution

    Lane 1 : Mouse brain lysate
    Lane 2 : Mouse heart lysate
    Lane 3 : Mouse kidney lysate
    Lane 4 : Mouse spleen lysate
    Lane 5 : NIH/3T3 (Mouse embryo fibroblast cells) whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

    Developed using the ECL technique.

    Predicted band size: 26 kDa
    Observed band size: 26 kDa


    Exposure time: 3 minutes


    Blocking and diluting buffer was 5% NFDM /TBST.

  • All lanes : Anti-Bcl-2 antibody [EPR17509] (ab182858) at 1/2000 dilution

    Lane 1 : Human fetal kidney lysate
    Lane 2 : Human fetal spleen lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

    Developed using the ECL technique.

    Predicted band size: 26 kDa
    Observed band size: 26 kDa


    Exposure time: 3 minutes


    Blocking and diluting buffer was 5% NFDM /TBST.

  • Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling Bcl-2 with ab182858 at 1/1000 followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500.

    Cytoplasm, nuclear membrane and nucleus staining on lymphocytes of Mouse spleen tissue is observed.

    Counter stained with Hematoxylin.
    Negative control: Used PBS instead of primary antibody followed by ab97051 at 1/500.

References

This product has been referenced in:
  • Yan X  et al. Lup-20(29)-en-3ß,28-di-yl-nitrooxy acetate affects MCF-7 proliferation through the crosstalk between apoptosis and autophagy in mitochondria. Cell Death Dis 9:241 (2018). Read more (PubMed: 29445224) »
  • Shi S  et al. Toxicity study of oxalicumone A, derived from a marine-derived fungus Penicillium oxalicum, in cultured renal epithelial cells. Mol Med Rep 15:2611-2619 (2017). Read more (PubMed: 28260084) »

See all 5 Publications for this product

Customer reviews and Q&As

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (Spleen, liver, kidney, pancreas.)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: EDTA
Permeabilization
No
Specification
Spleen, liver, kidney, pancreas.
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 2.5% · Temperature: 25°C
Fixative
Formaldehyde
Username

Abcam user community

Verified customer

Submitted May 23 2018

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Sign up