Astana Biomed Group, an authorized Abcam distributor in Central Asia

Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR3633(2)] to Calnexin - ER Membrane Marker
  • Suitable for: WB, IHC-P, Flow Cyt
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Overview

  • Product name

    Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker
    See all Calnexin primary antibodies

  • Description

    Rabbit monoclonal [EPR3633(2)] to Calnexin - ER Membrane Marker

  • Host species

    Rabbit

  • Specificity

    Recognizes ER membrane, mitochondria and cis-Golgi.

  • Tested Applications & Species

    Application Species
    Flow Cyt
    Human
    IHC-P
    Rat
    Human
    WB
    Human
    See all applications and species data

  • Immunogen

    Synthetic peptide within Human Calnexin aa 550 to the C-terminus (C terminal). The exact sequence is proprietary.
    Database link: P27824

  • Positive control

    • WB: WT HAP1, WT HEK-293T, HepG2, HeLa, A431, SH-SY5Y and THP1 cell lysate. IHC-P: Rat cardiac muscle; Human pancreas and kidney tissue. Flow Cyt: HeLa cells.

  • General notes

    Mouse: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

    References regarding specificity:

    Horner SM  et al. Mitochondrial-associated endoplasmic reticulum membranes (MAM) form innate immune synapses and are targeted by hepatitis C virus. Proc Natl Acad Sci U S A 108:14590-5 (2011). PubMed: 21844353

    Myhill N  et al. The subcellular distribution of calnexin is mediated by PACS-2. Mol Biol Cell 19:2777-88 (2008). PubMed: 18417615

    Yoshimura SI  et al. Direct targeting of cis-Golgi matrix proteins to the Golgi apparatus. J Cell Sci 114:4105-15 (2001). PubMed: 11739642

    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.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Properties

  • Form

    Liquid

  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Stable for 12 months at -20°C.

  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 0.05% BSA, 40% Glycerol, PBS
  • Concentration information loading...

  • Purity

    Protein A purified

  • Clonality

    Monoclonal

  • Clone number

    EPR3633(2)

  • Isotype

    IgG

  • Research areas

Images

  • Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    All lanes : Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615) at 1/1000 dilution

    Lane 1 : Wild-type HEK-293T cell lysate
    Lane 2 : CANX knockout HEK-293T cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 68 kDa
    Observed band size: 90 kDa
    why is the actual band size different from the predicted?



    Lanes 1- 2: Merged signal (red and green). Green - ab133615 observed at 90 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

     ab133615 was shown to react with Calnexin in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab255368 (knockout cell lysate ab263805) was used. Wild-type HEK-293T and CANX knockout HEK-293T 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. ab133615 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-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)

    Immunohistochemical staining of paraffin embedded rat cardiac muscle with purified ab133615 at a working dilution of 1 in 4000. The secondary antibody used is ab97051, a HRP goat anti-rabbit (H+L), at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

  • Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    All lanes : Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615) at 1/1000 dilution

    Lane 1 : Wild-type HAP1 cell lysate
    Lane 2 : Calnexin knockout HAP1 cell lysate
    Lane 3 : THP1 cell lysate
    Lane 4 : Raw264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 68 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab133615 observed at 80 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab133615 was shown to recognize Calnexin when Calnexin knockout samples were used, along with additional cross-reactive bands. Wild-type and Calnexin knockout samples were subjected to SDS-PAGE. ab133615 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. 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 1/10 000 dilution for 1 h at room temperature before imaging.

  • Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    All lanes : Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)

    Lane 1 : Wild-type HAP1 cell lysate
    Lane 2 : Calnexin knockout HAP1 cell lysate
    Lane 3 : THP1 cell lysate
    Lane 4 : Raw264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 68 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab133615 observed at 80 kDa. Red - loading control, ab8245, observed at 37 kDa.

    This western blot image is a comparison between ab133615 and a competitor's top cited rabbit polyclonal antibody.

  • Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615) at 1/1000 dilution (purified) + HepG2 (Human liver hepatocellular carcinoma cell line) cell lysate at 20 µg

    Secondary
    HRP goat anti-rabbit (H+L) at 1/1000 dilution

    Predicted band size: 68 kDa
    Observed band size: 90 kDa why is the actual band size different from the predicted?



    Blocking buffer: 5% NFDM/TBST

    Dilution buffer: 5% NFDM/TBST

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)

    Immunohistochemical staining of paraffin embedded human pancreas with purified ab133615 at a working dilution of 1 in 4000. The secondary antibody used is ab97051, a HRP goat anti-rabbit (H+L), at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

  • Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615) at 1/5000 dilution (purified) + HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 20 µg

    Secondary
    HRP goat anti-rabbit (H+L) at 1/1000 dilution

    Predicted band size: 68 kDa
    Observed band size: 90 kDa why is the actual band size different from the predicted?



    Blocking buffer: 5% NFDM/TBST

    Dilution buffer: 5% NFDM/TBST

  • Flow Cytometry - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    Flow Cytometry - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)

    Overlay histogram showing HeLa cells stained with unpurified ab133615 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab133615, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

  • Flow Cytometry - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    Flow Cytometry - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)

    Overlay histogram showing HeLa cells fixed in 80% methanol and stained with purified ab133615 at a dilution of 1 in 360 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 150. Rabbit monoclonal IgG was used as an isotype control (black line) and cells without incubation with antibody were used as a negative control (blue line).

  • Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    All lanes : Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615) at 1/1000 dilution (unpurified)

    Lane 1 : HepG2 (Human liver hepatocellular carcinoma cell line) cell lysate
    Lane 2 : A431 (Human epidermoid carcinoma cell line) cell lysate
    Lane 3 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) cell lysate
    Lane 4 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

    Predicted band size: 68 kDa
    Observed band size: 90 kDa why is the actual band size different from the predicted?

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)

    Immunohistochemical analysis of paraffin-embedded human kidney tissue labelled with unpurified ab133615 at 1/100 dilution.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)
    Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)

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