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    bcl-2-antibody-e17-bsa-and-azide-free-ab185002.pdf

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Cell Biology Apoptosis Intracellular Bcl2 Family
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)

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  • Certificate of Compliance
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Western blot - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
  • Immunoprecipitation - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
  • Western blot - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
  • Western blot - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
  • OI-RD Scanning - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
  • Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)

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 - BSA and Azide free
  • Suitable for: WB, IP, IHC-P
  • Knockout validated
  • Reacts with: Human

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Overview

  • Product name

    Anti-Bcl-2 antibody [E17] - BSA and Azide free
    See all Bcl-2 primary antibodies
  • Description

    Rabbit monoclonal [E17] to Bcl-2 - BSA and Azide free
  • Host species

    Rabbit
  • Tested Applications & Species

    Application Species
    IHC-P
    Human
    IP
    Human
    WB
    Human
    See all applications and species data
  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: HAP1, HeLa, THP-1 and MCF-7 cell lysates. IHC-P: Human DLBCL U2932, B-cell lymphoma, breast carcinoma and salivary gland tissue, and UM xenografts. IP: Jurkat cell lysate.
  • General notes

    Ab185002 is the carrier-free version of ab32124. This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    ab185002 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    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
    For more information see here.

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

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C. Do Not Freeze.
  • Dissociation constant (KD)

    KD = 3.00 x 10 -11 M
    Learn more about KD
  • Storage buffer

    pH: 7.20
    Constituent: PBS
  • Carrier free

    Yes
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    E17
  • Isotype

    IgG
  • Research areas

    • Cell Biology
    • Apoptosis
    • Intracellular
    • Bcl2 Family
    • Signal Transduction
    • Metabolism
    • Mitochondrial
    • Cancer
    • Invasion/microenvironment
    • Apoptosis
    • Bcl 2 family
    • Cancer
    • Oncoproteins/suppressors
    • Oncoproteins
    • Cell survival & death
    • Cancer
    • Tumor biomarkers
    • Oncoproteins
    • Cancer
    • Cancer Metabolism
    • Response to hypoxia
    • Cancer
    • Cancer Metabolism
    • Cellular metabolic process
    • Kits/ Lysates/ Other
    • Kits
    • ELISA Kits
    • ELISA Kits
    • Apoptosis marker and proteins ELISA kits
    • Metabolism
    • Pathways and Processes
    • Mitochondrial Metabolism
    • Mitochondrial markers
    • Metabolism
    • Pathways and Processes
    • Metabolism processes
    • Hypoxia
    • Metabolism
    • Pathways and Processes
    • Metabolism processes
    • Apoptosis
    • Cancer
    • Cell Death
    • Apoptosis
    • Apoptosis Markers
    • Bcl 2 family
    • Cancer
    • Cell Death
    • Apoptosis
    • Metabolism

Associated products

  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (ab96899)
  • Conjugation kits

    • PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
    • APC Conjugation Kit - Lightning-Link® (ab201807)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Positive Controls

    • Jurkat whole cell lysate (ab7899)
  • Recombinant Protein

    • Recombinant Human Bcl-2 protein (ab85156)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab185002 in the following tested applications.

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

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

Application Species
IHC-P
Human
IP
Human
WB
Human
Application Abreviews Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 26 kDa.

Please check the parent abID, ab32124, for a recommended dilution.

IP
Use at an assay dependent concentration.
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 26 kDa.

Please check the parent abID, ab32124, for a recommended dilution.

IP
Use at an assay dependent concentration.
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

  • Application notes
    Is unsuitable for Flow Cyt or ICC/IF.
  • 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.
    • Target information above from: UniProt accession P10415 The UniProt Consortium
      The Universal Protein Resource (UniProt) in 2010
      Nucleic Acids Res. 38:D142-D148 (2010) .

      Information by UniProt
    • Database links

      • Entrez Gene: 596 Human
      • Omim: 151430 Human
      • SwissProt: P10415 Human
      • Unigene: 150749 Human
      • 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
        • Protein phosphatase 1, regulatory subunit 50 antibody
        see all

      Images

      • Western blot - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
        Western blot - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
        All lanes : Anti-Bcl-2 antibody [E17] (ab32124) at 1/1000 dilution

        Lane 1 : Wild-type HeLa cell lysate
        Lane 2 : BCL2 HeLa knockout cell lysate

        Lysates/proteins at 20 µg per lane.

        Performed under reducing conditions.

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



        This data was developed using the same antibody clone in a different buffer formulation (ab32124).

          Lanes 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 Bcl-2 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.

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)This image is courtesy of an anonymous Abreview.

        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.

        Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

        This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32124).

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)Image from N?mati F et al. PLoS One. 2014;9(1):e80836. Fig 2.; doi: 10.1371/journal.pone.0080836.

        Bcl-2 expression determined by immunohistochemical analyses of the 4 human UM xenografts (between 3 to 5 tumors have been studied per condition).

        Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

        This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32124).

      • Immunoprecipitation - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
        Immunoprecipitation - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)

        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.

        This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32124).

      • Western blot - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
        Western blot - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
        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 kDa



        This data was developed using the same antibody clone in a different buffer formulation (ab32124).

        Lanes 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.

      • Western blot - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
        Western blot - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
        Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002) + MCF-7 (human breast carcinoma) whole cell lysate at 10 µg

        Secondary
        Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

        Predicted band size: 26 kDa


        Exposure time: 3 minutes


        Blocking buffer and concentration: 5% NFDM/TBST

        Diluting buffer and concentration: 5% NFDM/TBST

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)

        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.

        This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32124).

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)

        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling Bcl-2 with unpurified ab32124 at 1/200 dilution.

        Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

        This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32124).

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)

        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human B cell lymphoma tissue labelling Bcl-2 with unpurified ab32124.

        Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

        This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32124).

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)Image from Szyszko EA et al. Arthritis Res Ther. 2011 Jan 7;13(1):R2. Fig 5.; doi:10.1186/ar3220; 7 January 2011 Arthritis Research & Therapy 2011 13:R2.

        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.

        This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32124).

      • OI-RD Scanning - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
        OI-RD Scanning - Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)

        Equilibrium disassociation constant (KD)

        Click here to learn more about KD

        This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32124).

      • Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)
        Anti-Bcl-2 antibody [E17] - BSA and Azide free (ab185002)

      Protocols

      • Flow cytometry protocols
      • Immunoprecipitation protocols
      • Immunohistochemistry protocols
      • Western blot protocols

      Click here to view the general protocols

      Datasheets and documents

      • Datasheet

      Certificate of Compliance

      To download a Certificate of Compliance, please enter your Lot number below:

    • References (25)

      Publishing research using ab185002? Please let us know so that we can cite the reference in this datasheet.

      ab185002 has been referenced in 25 publications.

      • Shan H  et al. SNHG6 modulates oxidized low-density lipoprotein-induced endothelial cells injury through miR-135a-5p/ROCK in atherosclerosis. Cell Biosci 10:4 (2020). PubMed: 31921409
      • Ma Z  et al. LncRNA PCAT6 Accelerates the Progression and Chemoresistance of Cervical Cancer Through Up-Regulating ZEB1 by Sponging miR-543. Onco Targets Ther 13:1159-1170 (2020). PubMed: 32103984
      • Hu X  et al. LncRNA Oprm1 overexpression attenuates myocardial ischemia/reperfusion injury by increasing endogenous hydrogen sulfide via Oprm1/miR-30b-5p/CSE axis. Life Sci 254:117699 (2020). PubMed: 32437793
      • Shen XF  et al. MicroRNA-675-3p regulates IL-1ß-stimulated human chondrocyte apoptosis and cartilage degradation by targeting GNG5. Biochem Biophys Res Commun 527:458-465 (2020). PubMed: 32336544
      • Li H  et al. Circ_0072083 interference enhances growth-inhibiting effects of cisplatin in non-small-cell lung cancer cells via miR-545-3p/CBLL1 axis. Cancer Cell Int 20:78 (2020). PubMed: 32190002
      View all Publications for this product

      Customer reviews and Q&As

      Show All Reviews Q&A
      Submit a review Submit a question

      There are currently no Customer reviews or Questions for ab185002.
      Please use the links above to contact us or submit feedback about this product.

      Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
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