Validated using a knockout cell line
Recombinant
RabMAb

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

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

    Suitable for: WB, IHC-P, Flow Cytmore details
  • Species reactivity

    Reacts with: Rat, Human
  • Immunogen

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

  • Positive control

    • HepG2, A431, SH-SY5Y and HeLa cell lysates, Human kidney tissue
  • 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

    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.

    This product is a recombinant rabbit monoclonal antibody.

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

Applications

Our Abpromise guarantee covers the use of ab133615 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
WB 1/1000 - 1/5000. Detects a band of approximately 90 kDa (predicted molecular weight: 68 kDa).
IHC-P 1/4000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

For unpurified, use 1/100 - 1/250.

See IHC antigen retrieval protocols.

Flow Cyt 1/360.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Target

  • Function

    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.
  • Sequence similarities

    Belongs to the calreticulin family.
  • Cellular localization

    Endoplasmic reticulum membrane. Melanosome. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
  • Information by UniProt
  • Database links

  • Alternative names

    • 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

Images

  • 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 - 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.

  • 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.

  • 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 - 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.

  • Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615) at 1/1000 dilution (purified) + HepG2 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

  • 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.

  • Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615) at 1/5000 dilution (purified) + HeLa 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

  • 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.

  • 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).

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

    Lane 1 : HepG2 cell lysate
    Lane 2 : A431 cell lysate
    Lane 3 : SH-SY5Y cell lysate
    Lane 4 : HeLa 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?

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

References

This product has been referenced in:

  • Ouyang B  et al. Extracellular Vesicles From Human Urine-Derived Stem Cells Ameliorate Erectile Dysfunction in a Diabetic Rat Model by Delivering Proangiogenic MicroRNA. Sex Med 7:241-250 (2019). Read more (PubMed: 30910509) »
  • Chen J  et al. Aspirin inhibits hypoxia-mediated lung cancer cell stemness and exosome function. Pathol Res Pract N/A:152379 (2019). Read more (PubMed: 30878308) »
See all 4 Publications for this product

Customer reviews and Q&As

Application
Immunocytochemistry/ Immunofluorescence
Sample
Rat Cell (Rat Astrocytes)
Specification
Rat Astrocytes
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.3% TritonX in 0.1% PBS
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 24°C

Dr. Ruma Raha-Chowdhury

Verified customer

Submitted Dec 04 2012

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