使用敲除细胞株进行验证

Anti-Calnexin抗体[6F12BE10] (ab112995)

概述

  • 产品名称
    Anti-Calnexin抗体[6F12BE10]
    参阅全部 Calnexin 一抗
  • 描述
    小鼠单克隆抗体[6F12BE10] to Calnexin
  • 特异性
    Shotgun immunization of human HeLa cell lysates into mice. Targets were determined by mass spectrometry and validated by WB, ICC, ELISA pair and other techniques.
  • 经测试应用
    适用于: IP, In-Cell ELISA, Flow Cyt, ICC/IF, IHC-Pmore details
  • 种属反应性
    与反应: Human
  • 免疫原

    Hela cell fraction

  • 阳性对照
    • HeLa cells; HL60 cells and human fibroblasts.
  • 常规说明

    This antibody clone is manufactured by Abcam.

    This monoclonal antibody to calnexin has been knockout validated in WB and ICC/IF. The expected signal was observed in wild type cells and the signal was not seen in knockout cells.

性能

  • 形式
    Liquid
  • 存放说明
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Store at -20°C long term.
  • 存储溶液
    Preservative: 0.02% Sodium azide
    Constituent: 99.98% HBS
  • Concentration information loading...
  • 纯度
    >95% by SDS-PAGE
  • 纯化说明
    ab112995 was produced in vitro using hybridomas grown in serum-free medium, and then purified by biochemical fractionation. Monoclonal purity was near homogeneity as judged by SDS-PAGE.
  • 克隆
    单克隆
  • 克隆编号
    6F12BE10
  • 同种型
    IgG2b
  • 轻链类型
    kappa
  • 研究领域

应用

Our Abpromise guarantee covers the use of ab112995 in the following tested applications.

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

应用 Ab评论 说明
IP Use at an assay dependent concentration.
In-Cell ELISA Use a concentration of 1 µg/ml.
Flow Cyt Use a concentration of 1 µg/ml. ab170192-Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
ICC/IF Use a concentration of 0.5 µg/ml.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

靶标

  • 功能
    Calcium-binding protein that interacts with newly synthesized glycoproteins in the endoplasmic reticulum. It may act in assisting protein assembly and/or in the retention within the ER of unassembled protein subunits. It seems to play a major role in the quality control apparatus of the ER by the retention of incorrectly folded proteins.
  • 序列相似性
    Belongs to the calreticulin family.
  • 细胞定位
    Endoplasmic reticulum membrane. Melanosome. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
  • Information by UniProt
  • 数据库链接
  • 别名
    • Calnexin antibody
    • CALX_HUMAN antibody
    • CANX antibody
    • CNX antibody
    • FLJ26570 antibody
    • Histocompatibility complex class I antigen binding protein p88 antibody
    • IP90 antibody
    • Major histocompatibility complex class I antigen-binding protein p88 antibody
    • p90 antibody
    see all

Anti-Calnexin antibody [6F12BE10] 图像



  • Additional bands at : 80 kDa. We are unsure as to the identity of these extra bands.

    Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: Calnexin knockout HAP1 cell lysate (20 µg)
    Lane 3: THP1 cell lysate (20 µg)
    Lane 4: Raw264.7 cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab112995 observed at 80 kDa. Red - loading control, ab181602, observed at 37 kDa.

    ab112995 was shown to specifically react with Calnexin. Wild-type and Calnexin knockout samples were subjected to SDS-PAGE. ab112995 at a concentration of 1 µg/mL and ab181602 (loading control to GAPDH) diluted to 1/1000 were incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

  • ab112995 staining Calnexin (shown in green) in wild-type HAP1 cells (top panel) and CANX knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 min and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour. The cells were then incubated with ab112995 at 0.5 μg/ml and ab202272 at 1/250 dilution (alpha tubulin shown in red) overnight at +4°C, followed by a further incubation at room temperature for 1 hour with a goat secondary antibody to Mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • Immunoprecipitation with ab112995.
    Immunoprecipitation of Calnexin - ER membrane marker from HeLa cell lysate. The protein band runs around 90 kDa (predicted 68kDa) in tris-glycine SDS-PAGE. The identity of this protein was confirmed by mass spectrometry. This gel was stained with colloidal Coomassie blue G.
  • ab112995 at 5µg/ml staining Calnexin - ER membrane marker in Human fibroblasts cells by Immunocytochemistry (4% paraformaldehyde fixed and 0.1% Triton X-100 permeabilized) followed by Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h(green).
  • ab112995 at 1µg/ml staining Calnexin - ER membrane marker in HL60 cells fixed with MeOH by Flow Cytometry (blue). Isotype control antibody (red).
  • IHC image of ab112995 staining in human colon formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab112995, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Anti-Calnexin antibody [6F12BE10] (ab112995)参考文献

ab112995 has not yet been referenced specifically in any publications.

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