Recombinant
RabMAb

Recombinant Anti-CD33 antibody [EPR23051-101] (ab269456)

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Overview

  • Product name

    Anti-CD33 antibody [EPR23051-101]
    See all CD33 primary antibodies

  • Description

    Rabbit monoclonal [EPR23051-101] to CD33

  • Host species

    Rabbit

  • Tested applications

    Suitable for: IHC-P, ICC/IF, IP, WBmore details
    Unsuitable for: Flow Cyt

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Synthetic peptide within Human CD33 aa 250-350. The exact sequence is proprietary.
    Database link: P20138

  • Positive control

    • WB: U937 and THP-1 whole cell lysates; Human liver lysate. IHC-P: Human liver, Human Hodgkiin's lymphoma and Human clear cell renal cell carcinoma tissue . ICC/IF: THP-1 cells. IP: THP-1 cells.

  • General notes

    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

Applications

Our Abpromise guarantee covers the use of ab269456 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
IHC-P 1/200. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF 1/50.
IP 1/30.
WB 1/1000. Predicted molecular weight: 40 kDa.
  • Application notes
    Is unsuitable for Flow Cyt.

    • Target

    • Function

      Putative adhesion molecule of myelomonocytic-derived cells that mediates sialic-acid dependent binding to cells. Preferentially binds to alpha-2,6-linked sialic acid. The sialic acid recognition site may be masked by cis interactions with sialic acids on the same cell surface. In the immune response, may act as an inhibitory receptor upon ligand induced tyrosine phosphorylation by recruiting cytoplasmic phosphatase(s) via their SH2 domain(s) that block signal transduction through dephosphorylation of signaling molecules. Induces apoptosis in acute myeloid leukemia (in vitro).

    • Tissue specificity

      Monocytic/myeloid lineage cells.

    • Sequence similarities

      Belongs to the immunoglobulin superfamily. SIGLEC (sialic acid binding Ig-like lectin) family.
      Contains 1 Ig-like C2-type (immunoglobulin-like) domain.
      Contains 1 Ig-like V-type (immunoglobulin-like) domain.

    • Domain

      Contains 2 copies of a cytoplasmic motif that is referred to as the immunoreceptor tyrosine-based inhibitor motif (ITIM). This motif is involved in modulation of cellular responses. The phosphorylated ITIM motif can bind the SH2 domain of several SH2-containing phosphatases.

    • Post-translational
      modifications

      Phosphorylation of Tyr-340 is involved in binding to PTPN6 and PTPN11. Phosphorylation of Tyr-358 is involved in binding to PTPN6.

    • Cellular localization

      Cell membrane.
    • Information by UniProt

    • Database links

      • Alternative names

        • CD 33 antibody
        • CD33 antibody
        • CD33 antigen (gp67) antibody
        • CD33 antigen antibody
        • CD33 molecule antibody
        • CD33_HUMAN antibody
        • FLJ00391 antibody
        • gp67 antibody
        • My9 antibody
        • Myeloid cell surface antigen CD33 antibody
        • Myeloid cell surface antigen CD33 precursor antibody
        • Myeloid differentiation antigen CD33 antibody
        • p67 antibody
        • Sialic acid binding Ig like lectin 3 antibody
        • Sialic acid binding immunoglobulin like lectin 3 antibody
        • Sialic acid-binding Ig-like lectin 3 antibody
        • SIGLEC 3 antibody
        • Siglec-3 antibody
        • SIGLEC3 antibody
        see all

      Images

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD33 antibody [EPR23051-101] (ab269456)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD33 antibody [EPR23051-101] (ab269456)

        Immunohistochemical analysis of paraffin-embedded Human Hodgkin's lymphoma tissue labeling CD33 with ab269456 at 1/200 dilution (2.96 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous staining on human Hodgkin's lymphoma.

        The section was incubated with ab269456 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

        Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

        Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

      • Immunoprecipitation - Anti-CD33 antibody [EPR23051-101] (ab269456)
        Immunoprecipitation - Anti-CD33 antibody [EPR23051-101] (ab269456)

        CD33 was immunoprecipitated from 0.35 mg THP-1 (Human monocytic leukemia monocyte) whole cell lysate with ab269456 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab269456 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

        Lane 1: THP-1 whole cell lysate 10ug.

        Lane 2: ab269456 IP in THP-1 whole cell lysate.

        Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab269456 in THP-1 whole cell lysate.

        Blocking and dilution buffer and concentration: 5% NFDM/TBST.

        Exposure time: 23 seconds.

         

      • Immunocytochemistry/ Immunofluorescence - Anti-CD33 antibody [EPR23051-101] (ab269456)
        Immunocytochemistry/ Immunofluorescence - Anti-CD33 antibody [EPR23051-101] (ab269456)

        Immunofluorescent analysis of 100% methanol-fixed THP-1 (human monocytic leukemia monocyte) and Jurkat (human T cell leukemia T lymphocyte) cells labeling CD33 with ab26946 at 1/50 dilution, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution (Green). Confocal image showing membranous staining in THP-1 cells. The nuclear counter stain is DAPI (Blue). Tubilin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1/200 dilution (Red).

        Control: Used PBS instead of primary antibody, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.

        Negative control: Jurkat (PMID: 30519686).

         

      • Western blot - Anti-CD33 antibody [EPR23051-101] (ab269456)
        Western blot - Anti-CD33 antibody [EPR23051-101] (ab269456)
        All lanes : Anti-CD33 antibody [EPR23051-101] (ab269456) at 1/1000 dilution

        Lane 1 : U937 (human histiocytic lymphoma monocyte), whole cell lysate
        Lane 2 : THP-1 (human monocytic leukemia monocyte), whole cell lysate
        Lane 3 : Jurkat (human T cell leukemia T lymphocyte), whole cell lysate

        Lysates/proteins at 20 µg per lane.

        Secondary
        All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

        Predicted band size: 40 kDa
        Observed band size: 67/75 kDa why is the actual band size different from the predicted?



        Blocking and dilution buffer: 5% NFDM/TBST.

        Exposure time: 70 seconds.

        The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID:16380601, 30519686).

        Negative Control:Jurkat (PMID:30519686).

      • Western blot - Anti-CD33 antibody [EPR23051-101] (ab269456)
        Western blot - Anti-CD33 antibody [EPR23051-101] (ab269456)
        All lanes : Anti-CD33 antibody [EPR23051-101] (ab269456) at 1/1000 dilution

        Lane 1 : Human liver tissue lysate
        Lane 2 : Human skeletal muscle tissue lysate

        Lysates/proteins at 20 µg per lane.

        Secondary
        All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

        Predicted band size: 40 kDa
        Observed band size: 75 kDa why is the actual band size different from the predicted?



        Blocking and dilution buffer: 5% NFDM/TBST.

        Exposure time: 70 seconds.

        The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID:16380601, 30519686).

        Negative control:Human skeletal muscle.

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD33 antibody [EPR23051-101] (ab269456)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD33 antibody [EPR23051-101] (ab269456)

        Immunohistochemical analysis of paraffin-embedded Human clear cell renal cell carcinoma tissue labeling CD33 with ab269456 at 1/200 dilution (2.96 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous staining on infiltrated myeloid cells of human clear cell renal cell carcinoma The section was incubated with ab269456 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

        Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

        Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD33 antibody [EPR23051-101] (ab269456)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD33 antibody [EPR23051-101] (ab269456)

        Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling CD33 with ab269456 at 1/200 dilution (2.96 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous staining on mainly Kuffer cells of human liver (PMID:25721896). The section was incubated with ab269456 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

        Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

        Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD33 antibody [EPR23051-101] (ab269456)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD33 antibody [EPR23051-101] (ab269456)

        Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling CD33 with ab269456 at 1/200 dilution (2.96 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Counterstained with Hematoxylin.

        Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

        Negative control：Human skeletal muscle.

        The section was incubated with ab269456 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument

        Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

      Protocols

      To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

      Click here to view the general protocols

      References

      Publishing research using ab269456? Please let us know so that we can cite the reference in this datasheet.

      ab269456 has not yet been referenced specifically in any publications.

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