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使用敲除细胞株进行验证重组RabMAb

重组Anti-CD73抗体[RM2050] - BSA and Azide free (ab317463)

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Western blot - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
  • Western blot - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
  • Immunocytochemistry/ Immunofluorescence - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
  • Immunocytochemistry/ Immunofluorescence - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
  • Flow Cytometry - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
  • Flow Cytometry - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
  • Flow Cytometry - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
  • Flow Cytometry - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
  • Immunoprecipitation - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
  • Immunoprecipitation - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
  • Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit recombinant multiclonal [RM2050] to CD73 - BSA and Azide free
  • Suitable for: IP, ICC/IF, Flow Cyt, IHC-P, WB
  • Knockout validated
  • Reacts with: Mouse, Human

Conjugates logo Related conjugates and formulations

Unconjugated

概述

  • 产品名称

    Anti-CD73抗体[RM2050] - BSA and Azide free
    参阅全部 CD73 一抗

  • 描述

    兔重组multiclonal [RM2050] to CD73 - BSA and Azide free

  • 宿主

    Rabbit

  • 特异性

    Unsuitable for mouse IHC.

  • 经测试应用

    适用于: IP, ICC/IF, Flow Cyt, IHC-P, WBmore details

  • 种属反应性

    与反应: Mouse, Human
    不与反应: Rat

  • 免疫原

    This product was produced with the following immunogens:
    Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.

    Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.

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

  • 阳性对照

    • WB: U-87 MG whole cell, A375 whole cell, Wild-type A431 whole cell, Human spleen tissue and 4T1 whole cell lysates. IHC-P: Human spleen, Human liver cancer and Human stomach tissues. ICC/IF: A431 and 4T1 cells. Flow Cyt: 4T1, Parental A431, HeLa, U-87 MG and Mouse spleen cells. IP: ab317462 IP in U-87 MG and 4T1 whole cell lysates.

  • 常规说明

    ab317463 is the carrier-free version of ab317462

    Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.

    This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.

    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.

    This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. 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.

性能

应用

The Abpromise guarantee

Abpromise™承诺保证使用ab317463于以下的经测试应用

“应用说明”部分 下显示的仅为推荐的起始稀释度；实际最佳的稀释度/浓度应由使用者检定。

应用 Ab评论 说明
IP
Use at an assay dependent concentration.
ICC/IF
Use at an assay dependent concentration.
Flow Cyt
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.
WB
Use at an assay dependent concentration.
说明
IP
Use at an assay dependent concentration.
ICC/IF
Use at an assay dependent concentration.
Flow Cyt
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.
WB
Use at an assay dependent concentration.

靶标

  • 功能

    Hydrolyzes extracellular nucleotides into membrane permeable nucleosides.

  • 疾病相关

    Defects in NT5E are the cause of calcification of joints and arteries (CAJA) [MIM:211800]. A condition characterized by adult-onset calcification of the lower extremity arteries, including the iliac, femoral and tibial arteries, and hand and foot capsule joints. Age of onset has been reported as early as the second decade of life, usually involving intense joint pain or calcification in the hands.

  • 序列相似性

    Belongs to the 5'-nucleotidase family.

  • 细胞定位

    Cell membrane.
  • Information by UniProt

  • 数据库链接

    • 别名

      • 5' NT antibody
      • 5' nucleotidase (CD73) antibody
      • 5' nucleotidase precursor antibody
      • 5' nucleotidase, ecto antibody
      • 5' nucleotidase, ecto (CD73) antibody
      • 5'-NT antibody
      • 5'-nucleotidase antibody
      • 5NTD_HUMAN antibody
      • CD73 antibody
      • CD73 antigen antibody
      • E5NT antibody
      • Ecto 5' nucleotidase antibody
      • Ecto-5'-nucleotidase antibody
      • eN antibody
      • eNT antibody
      • NT antibody
      • NT5 antibody
      • NT5E antibody
      • NTE antibody
      • Purine 5 Prime Nucleotidase antibody
      see all

    图片

    • Western blot - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
      Western blot - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
      All lanes : Anti-CD73 antibody [RM2050] (ab317462) at 1/1000 dilution

      Lane 1 : U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate
      Lane 2 : A375 (human malignant melanoma epithelial cell) whole cell lysate
      Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
      Lane 4 : Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate
      Lane 5 : Wild-type A431 (human epidermoid carcinoma epithelial cell) whole cell lysate
      Lane 6 : CD73 knockout A431 whole cell lysate
      Lane 7 : Human spleen tissue lysate

      Lysates/proteins at 40 µg per lane.

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

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


      Exposure time: 180 seconds


      This data was developed using ab317462, the same antibody clone in a different buffer formulation.

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

      Negative control: HeLa and Raji(PMID: 8566797)

      In Western blot, ab317462 was shown to bind specifically to CD73. A band was observed at 75 kDa in wild-type A431 cell lysates (lane 5) whereas loss of signal was observed in the CD73 knockout cell line (lane 6, ab261895/knockout cell lysate ab261704).

      In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

    • Western blot - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
      Western blot - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
      All lanes : Anti-CD73 antibody [RM2050] (ab317462) at 1/1000 dilution

      Lane 1 : 4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate
      Lane 2 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate

      Lysates/proteins at 40 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

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


      Exposure time: 180 seconds


      This data was developed using ab317462, the same antibody clone in a different buffer formulation.

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

      Negative control: RAW264.7(PMID:28060378)

      In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)

      This data was developed using ab317462, the same antibody clone in a different buffer formulation.

      Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling CD73 with ab317462 at 1/100 (4.97 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

      Positive staining on human spleen. The section was incubated with ab317462 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

      Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)

      This data was developed using ab317462, the same antibody clone in a different buffer formulation.

      Immunohistochemical analysis of paraffin-embedded Human liver cancer tissue labeling CD73 with ab317462 at 1/100 (4.97 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

      Positive staining on human liver cancer (PMID: 30971294). The section was incubated with ab317462 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

      Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)

      This data was developed using ab317462, the same antibody clone in a different buffer formulation.

      Immunohistochemical analysis of paraffin-embedded Human stomach tissue labeling CD73 with ab317462 at 1/100 (4.97 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

      Low expression: Weakly positive staining on blood vessels of human stomach. The section was incubated with ab317462 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

      Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

    • Immunocytochemistry/ Immunofluorescence - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
      Immunocytochemistry/ Immunofluorescence - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)

      This data was developed using ab317462, the same antibody clone in a different buffer formulation.

      Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NT5E KO A431(human epidermoid carcinoma epithelial cell) cells labelling CD73 with ab317462 at 1/500 (0.994 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/mL dilution (Green).

      Confocal image showing membrane and cytoplasmic staining in parental A431 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

      ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

      Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

    • Immunocytochemistry/ Immunofluorescence - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
      Immunocytochemistry/ Immunofluorescence - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)

      This data was developed using ab317462, the same antibody clone in a different buffer formulation.

      Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 4T1 (mouse mammary gland carcinoma epithelial cell) cells labelling CD73 with ab317462 at 1/500 (0.994 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/mL dilution (Green).

      Confocal image showing membrane and cytoplasmic staining in 4T1 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
      Negative control: RAW 264.7 (PMID: 28060378)
      Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

      ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

      Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

    • Flow Cytometry - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
      Flow Cytometry - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)

      This data was developed using ab317462, the same antibody clone in a different buffer formulation.

      Flow cytometric analysis of RAW 264.7(mouse Abelson murine leukemia virus-induced tumor macrophage, Left) / 4T1 (mouse mammary gland carcinoma epithelial cell, Right) cells labelling CD73 with ab317462 at 1/50 dilution (1 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

      Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

      Negative control: RAW 264.7(PMID:28060378)
      Gated on viable cell.

    • Flow Cytometry - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
      Flow Cytometry - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)

      This data was developed using ab317462, the same antibody clone in a different buffer formulation.

      Flow cytometric analysis of CD73 knockout A431(huam CD73 knock out human epidermoid carcinoma epithelial cell, Left) / Parental A431(Right) cells labelling CD73 with ab317462 at 1/500 dilution (0.1 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

      Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

      Gated on viable cell.

    • Flow Cytometry - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
      Flow Cytometry - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)

      This data was developed using ab317462, the same antibody clone in a different buffer formulation.

      Flow cytometric analysis of HeLa(human cervical adenocarcinoma epithelial cell, Left) / U-87 MG(human glioblastoma-astrocytoma epithelial cell, Right) cells labelling CD73 with ab317462 at 1/5000 dilution (0.01 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

      Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

      Low expression: HeLa (PMID: 8566797)
      Gated on viable cell.

    • Flow Cytometry - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
      Flow Cytometry - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)

      This data was developed using ab317462, the same antibody clone in a different buffer formulation.

      Flow cytometric analysis of Mouse spleen cells labelling CD73 with ab317462 at 1/50 dilution (1 ug)/Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

      Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

      Mouse spleen cell are co-stained with CD3 conjugated Alexa Fluor®647.
      Gated on viable cell.

    • Immunoprecipitation - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
      Immunoprecipitation - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)

      This data was developed using ab317462, the same antibody clone in a different buffer formulation.

      CD73 was immunoprecipitated from 0.35 mg U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate with ab317462 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317462 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

      Lane 1: U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate
      Lane 2: ab317462 IP in U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate
      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab317462 in U-87 MG whole cell lysate

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

      Exposure time: 32 seconds.

    • Immunoprecipitation - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
      Immunoprecipitation - Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)

      This data was developed using ab317462, the same antibody clone in a different buffer formulation.

      CD73 was immunoprecipitated from 0.35 mg 4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate with ab317462 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317462 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

      Lane 1: 4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate
      Lane 2: ab317462 IP in 4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate
      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab317462 in 4T1 whole cell lysate

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

      Exposure time: 180 seconds.

    • Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)
      Anti-CD73 antibody [RM2050] - BSA and Azide free (ab317463)

    实验方案

    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

    文献 (0)

    发表研究结果有使用 ab317463？请让我们知道，以便我们可以引用本数据表中的参考文章。

    ab317463 尚未被引用在任何文献中。

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