Overview

  • Product name
  • Description
    Rabbit polyclonal to Calnexin
  • Host species
    Rabbit
  • Specificity
    Recognizes ER membrane, mitochondria and cis-Golgi
  • Tested applications
    Suitable for: WB, ICC/IF, IHC-P, IP, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Common marmoset
    Predicted to work with: Dog
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 550 to the C-terminus of Human Calnexin.

    Read Abcam's proprietary immunogen policy (Peptide available as ab23379.)

  • Positive control
    • WB: HeLa, MCF-7, NIH/3T3, MEF1 and PC-12 whole cell lysates, mouse brain, liver, heart, kidney, pancreas, testis, skeletal muscle, spinal cord and ovary and rat brain, liver, heart and kidney tissue lysates. Wild-type HAP1 whole cell lysate. ICC/IF: HeLa and wild-type HAP1 cells. IP: HeLa whole cell lysate.
  • General notes

    Abcam recommended secondaries - Goat Anti-Rabbit HRP (ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (ab150077).

    See other anti-rabbit secondary antibodies that can be used with this 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 or -80°C. Avoid freeze / thaw cycle. Store In the Dark.
  • Storage buffer
    Preservative: 0.02% Sodium Azide
    Constituents: 1% BSA, PBS, pH 7.4
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab22595 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 Use a concentration of 1 µg/ml. Detects a band of approximately 75 kDa (predicted molecular weight: 90 kDa).
ICC/IF Use a concentration of 1 µg/ml.
IHC-P Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.

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

  •  

    Subcellular distribution of Smn and hnRNP R in isolated embryonic motoneurons.

    Lentiviral knockdown of hnRNP R led to a dose-dependent reduction of hnRNP R levels. Calnexin and Smn protein were not altered significantly.

    Primary motoneurons or E18 spinal cord tissue, respectively, were lysed with cytosolic and nuclear fractionation buffer, solubilized in Laemmli buffer and boiled for 10 minutes at 99°C. Proteins were then subjected to SDS-PAGE, blotted onto PVDF membrane, incubated with the corresponding antibodies, including ab22595.

     

  • Lane 1: Wild type HAP1 whole cell lysate (20 µg)
    Lane 2: empty lane
    Lane 3: CANX knockout HAP1 whole cell lysate (20 µg)
    Lane 4: empty lane
    Lanes 1 - 4: Merged signal (red and green). Green - ab22595 observed at 80 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab22595 was shown to specifically react with CANX (Calnexin) in wildtype cells as signal was lost in CANX (Calnexin) knockout cells. Wild-type and eCANX (Calnexin) knockout samples were subjected to SDS-PAGE. ab22595 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1 dilution and 1/10,000 dilution respectively.

    Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

  • ab22595 staining Calnexin in wild-type HAP1 cells (top panel) and CANX knockout HAP1 cells (bottom panel).

    The cells were fixed with 4% formaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes 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 ab22595 at 1 μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1 hour with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green).

    Nuclear DNA was labeled in blue with DAPI.

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

  • ab22595 staining Calnexin in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

    The cells were fixed with 100% methanol for 5 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes 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 ab22595 at 1 μg/ml and ab7291 at 1 μg/ml overnight at +4°C, followed by a further incubation at room temperature for 1 hour with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to Mouse IgG (Alexa Fluor® 594) (ab150120).

    Nuclear DNA was labeled in blue with DAPI.

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

  • Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: Calnexin knockout HAP1 cell lysate (20 µg)

    Lanes 1 - 2: Merged signal (red and green). Green - ab22595 observed at 80 kDa. Red - loading control, ab8245, observed at 37 kDa.

    This western blot image is a comparison between ab22595 and a competitor's top cited rabbit polyclonal antibody.

  • All lanes : Anti-Calnexin antibody (ab22595) at 1/250 dilution

    Lane 1 : NIH 3T3 whole cell lysate (ab7179)
    Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab46770)
    Lane 3 : Brain (Mouse) Tissue Lysate (ab27253)
    Lane 4 : Liver (Mouse) Tissue Lysate (ab7935)
    Lane 5 : Heart (Mouse) Tissue Lysate (ab27255)
    Lane 6 : Kidney (Mouse) Tissue Lysate (ab27254)
    Lane 7 : Mouse pancreas tissue lysate - total protein (ab29363)
    Lane 8 : Testis (Mouse) Tissue Lysate - normal tissue (ab4027)
    Lane 9 : Mouse skeletal muscle tissue lysate - total protein (ab29711)
    Lane 10 : Spinal Cord (Mouse) Tissue Lysate (ab50253)
    Lane 11 : Ovary (Mouse) Tissue Lysate (ab35808)
    Lane 12 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate (ab50957)
    Lane 13 : Brain (Rat) Tissue Lysate (ab7942)
    Lane 14 : Liver (Rat) Tissue Lysate (ab27256)
    Lane 15 : Heart (Rat) Tissue Lysate (ab7940)
    Lane 16 : Kidney (Rat) Whole Cell Lysate - normal tissue (ab29480)

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 90 kDa
    Observed band size: 80 kDa why is the actual band size different from the predicted?

  • Kidney cortex using ab22595 shows clear cytoplasmic staining patterns.

    The visceral cells of the glomerular tuft (podocytes) are strongly stained (indicated by red arrowheads).

    Distal convoluted tubular cells are generally moderately positive (with exceptions that are strongly positive).

    However, most of the cells that line the proximal convoluted tubules (indicated by green arrowheads) are strongly positive.

    See Abreview

  • All lanes : Anti-Calnexin antibody (ab22595) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) whole cell lysate
    Lane 2 : U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate
    Lane 3 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
    Lane 4 : HeLa whole cell lysate with Human Calnexin peptide (ab23379) at 1 µg/ml
    Lane 5 : U-2 OS whole cell lysate with Human Calnexin peptide (ab23379) at 1 µg/ml
    Lane 6 : MCF7 whole cell lysate with Human Calnexin peptide (ab23379) at 1 µg/ml

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG (Alexa Fluor® 680) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 90 kDa
    Observed band size: 75 kDa why is the actual band size different from the predicted?



    Recent batches of ab22595 (AP217379 and AP151845) detect a band of ~ 75 kDa in HeLa, U-2 OS and MCF7 lysates. This band is completely blocked by the immunizing peptide so we believe this represents Calnexin. Moreoever, a band of the same size is detected by other Calnexin antibodies tested.

     

  • Calnexin was immunoprecipitated using 0.5 mg HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell extract, 5 µg of Rabbit polyclonal to Calnexin - ER membrane marker and 50 µl of protein G magnetic beads (+).

    No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10 minutes, HeLa whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10 minutes under agitation.
    Proteins were eluted by addition of 40 µl SDS loading buffer and incubated for 10 minutes at 70oC; 10 µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab22595.
    Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
    Band: 80kDa: Calnexin - ER membrane marker.

References

This product has been referenced in:
  • Kathayat RS  et al. Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes. Nat Commun 9:334 (2018). Read more (PubMed: 29362370) »
  • Rödiger M  et al. Adiponectin release and insulin receptor targeting share trans-Golgi-dependent endosomal trafficking routes. Mol Metab 8:167-179 (2018). WB ; Human . Read more (PubMed: 29203237) »
See all 113 Publications for this product

Customer reviews and Q&As

1-10 of 32 Abreviews or Q&A

Application
Western blot
Sample
Sheep Cell lysate - other (total protein, mitochondria and ER isolates)
Gel Running Conditions
Reduced Denaturing (10% PAG)
Loading amount
10 µg
Specification
total protein, mitochondria and ER isolates
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 23°C

Miss. Barbara Makela

Verified customer

Submitted Mar 08 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HEK 293t, HFF)
Permeabilization
Yes - 0.1% TX-100, 0.01% SDS in 1xPBS
Specification
HEK 293t, HFF
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 10% · Temperature: RT°C
Fixative
Formaldehyde

Miss. Kim JEONG-JIN

Verified customer

Submitted Feb 20 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (primary keratinocyte)
Permeabilization
Yes - 0.4% TritonX100
Specification
primary keratinocyte
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 12% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Sep 05 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (face skin)
Permeabilization
Yes - 0.4% TritonX100
Specification
face skin
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Acetone

Abcam user community

Verified customer

Submitted Sep 01 2016

Application
Immunocytochemistry
Sample
Rat Cultured Cells (primary Hippocampal neuron)
Permeabilization
Yes - 0.2% Triton X-100 in PBS
Specification
primary Hippocampal neuron
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 23°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Mar 11 2016

Application
Western blot
Sample
Rat Cell lysate - whole cell (RBL-2H3 cell)
Gel Running Conditions
Reduced Denaturing
Loading amount
10 µg
Specification
RBL-2H3 cell
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Sep 11 2015

Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293T cells)
Gel Running Conditions
Reduced Denaturing
Loading amount
10 µg
Specification
HEK293T cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Jun 19 2015

Application
Western blot
Loading amount
10000 cells
Gel Running Conditions
Reduced Denaturing (4-12% gradient)
Sample
Human Cell lysate - whole cell (Huh7 liver)
Specification
Huh7 liver
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Feb 05 2015

Application
Western blot
Loading amount
10 µg
Gel Running Conditions
Reduced Denaturing (16%)
Sample
Mouse Cell lysate - whole cell (Primary bone marrow derived macrophage)
Specification
Primary bone marrow derived macrophage
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Oct 16 2014

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Sample
Human Cell (ovarian)
Specification
ovarian
Permeabilization
Yes - 0.1% triton X for 5 min at RT
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Aug 25 2014

1-10 of 32 Abreviews or Q&A

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