Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Calreticulin antibody [EPR3924] - ER Marker (ab92516)

Overview

  • Product name

    Anti-Calreticulin antibody [EPR3924] - ER Marker
    See all Calreticulin primary antibodies
  • Description

    Rabbit monoclonal [EPR3924] to Calreticulin - ER Marker
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-P, Flow Cyt, ICC/IFmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human, Monkey
  • Immunogen

    Synthetic peptide within Human Calreticulin aa 50-150. The exact sequence is proprietary.
    (Peptide available as ab180826)

  • Positive control

    • WB: SH-SY5Y, HL-60, HepG2, HeLa, Fetal kidney and Fetal brain lysates; Human kidney tissue; Mouse and Rat brain lysates. ICC/IF: HAP1 cells (HAP1-CALR as negative cell line) IHC-P: Human colon, kidney, liver, placenta, stomach, breast carcinoma and Papillary carcinoma of thyroid gland tissues; Mouse liver and Rat lung tissues.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    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: 9% PBS, 40% Glycerol, 0.05% BSA, 50% Tissue culture supernatant
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR3924
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab92516 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 - 1/10000. Predicted molecular weight: 48 kDa.
IHC-P 1/250 - 1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

The use of a HRP/AP polymerized secondary antibody is recommended for enhanced staining.

Flow Cyt 1/10 - 1/100.

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

ICC/IF 1/500.

Target

  • Function

    Molecular calcium-binding chaperone promoting folding, oligomeric assembly and quality control in the ER via the calreticulin/calnexin cycle. This lectin interacts transiently with almost all of the monoglucosylated glycoproteins that are synthesized in the ER. Interacts with the DNA-binding domain of NR3C1 and mediates its nuclear export.
  • Sequence similarities

    Belongs to the calreticulin family.
  • Domain

    Can be divided into a N-terminal globular domain, a proline-rich P-domain forming an elongated arm-like structure and a C-terminal acidic domain. The P-domain binds one molecule of calcium with high affinity, whereas the acidic C-domain binds multiple calcium ions with low affinity.
    The interaction with glycans occurs through a binding site in the globular lectin domain.
    The zinc binding sites are localized to the N-domain.
    Associates with PDIA3 through the tip of the extended arm formed by the P-domain.
  • Cellular localization

    Endoplasmic reticulum lumen. Cytoplasm > cytosol. Secreted > extracellular space > extracellular matrix. Cell surface. Also found in cell surface (T cells), cytosol and extracellular matrix. Associated with the lytic granules in the cytolytic T-lymphocytes.
  • Information by UniProt
  • Database links

  • Alternative names

    • Autoantigen RO antibody
    • CALR antibody
    • CALR protein antibody
    • CALR_HUMAN antibody
    • Calregulin antibody
    • Calreticulin antibody
    • cC1qR antibody
    • CRP55 antibody
    • CRT antibody
    • CRTC antibody
    • Endoplasmic reticulum resident protein 60 antibody
    • Epididymis secretory sperm binding protein Li 99n antibody
    • ERp60 antibody
    • FLJ26680 antibody
    • grp60 antibody
    • HACBP antibody
    • HEL S 99n antibody
    • RO antibody
    • Sicca syndrome antigen A (autoantigen Ro; calreticulin) antibody
    • Sicca syndrome antigen A antibody
    • SSA antibody
    see all

Images

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

    Lane 2: Calreticulin knockout HAP1 cell lysate (20 µg)

    Lane 3: HeLa cell lysate (20 µg)

    Lane 4: NIH3T3 cell lysate (20 µg)

    Lanes 1 - 4: Merged signal (red and green). Green - ab92516 observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab92516 was shown to specifically react with Calreticulin when Calreticulin knockout samples were used. Wild-type and Calreticulin knockout samples were subjected to SDS-PAGE. ab92516 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

  • ab92516 staining Calreticulin in wild-type HAP1 cells (top panel) and CALR knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 1h. The cells were then incubated with ab92516 at 1/500 and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) 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).

  • Immunohistochemical analysis of paraffin-embedded Rat lung tissue labeling Calreticulin  with ab92516, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on rat lung.The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

  • All lanes : Anti-Calreticulin antibody [EPR3924] - ER Marker (ab92516) at 1/10000 dilution

    Lane 1 : HepG2 (Human hepatocellular carcinoma epithelial cell) Whole cell lysates
    Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
    Lane 3 : Mouse brain lysates
    Lane 4 : Rat brain lysates
    Lane 5 : COS-1 (African green monkey kidney fibroblast-like) whole cell lysates

    Lysates/proteins at 15 µg per lane.

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

    Predicted band size: 48 kDa
    Observed band size: 55 kDa why is the actual band size different from the predicted?



    Blocking/Diluting buffer and concentration: 5% NFDM/TBST

    Exposure time: Lane 1 to 3: 10 seconds; Lane 4 and 5: 130 seconds

  • Overlay histogram showing HAP1 wildtype (green line) and HAP1-CALR knockout cells (red line) stained with ab92516. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab92516, 1µg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) preadsorbed (ab150081) at 1/2000 dilution for 30 min at 22°C.

    A rabbit IgG isotype control antibody  (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-CALR knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

    Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

  • Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Calreticulin  with ab92516, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on mouse liver.The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

  • Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling Calreticulin with ab92516, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining human breast carcinoma.The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

  • Formalin-fixed, paraffin-embedded normal human colon tissue stained for Calreticulin using ab92516 at 1/250 dilution in immunohistochemical analysis.

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

  • Immunocytochemistry/Immunofluorescence analysis of HT-29 (human colorectal adenocarcinoma) labelling Calreticulin with purified ab92516 at 1/500. Cells were fixed with 100% methanol. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Ab150077). Nuclei counterstained with DAPI (blue).

    Control: PBS only

  • Overlay histogram showing HeLa cells stained with ab92516 (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 (ab92516, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • All lanes : Anti-Calreticulin antibody [EPR3924] - ER Marker (ab92516) at 1/1000 dilution

    Lane 1 : SH-SY5Y cell lysate
    Lane 2 : HL-60 cell lysate
    Lane 3 : HepG2 cell lysate
    Lane 4 : HeLa cell lysate
    Lane 5 : Human fetal kidney lysate
    Lane 6 : Human fetal brain lysate

    Lysates/proteins at 10 µg per lane.

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

    Predicted band size: 48 kDa

  • ab92516, at 1/250 dilution, staining Calreticulin in paraffin embedded Human kidney tissue by Immunohistochemistry.

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

  • ab92516 showing negative staining in Normal human heart tissue.

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

  • Formalin-fixed, paraffin-embedded normal human liver tissue stained for Calreticulin using ab92516 at 1/250 dilution in immunohistochemical analysis.

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

  • Formalin-fixed, paraffin-embedded normal human placenta tissue stained for Calreticulin using ab92516 at 1/250 dilution in immunohistochemical analysis.



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

  • Formalin-fixed, paraffin-embedded normal human stomach tissue stained for Calreticulin using ab92516 at 1/250 dilution in immunohistochemical analysis.

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

  • Formalin-fixed, paraffin-embedded Papillary carcinoma of human thyroid gland tissue.stained for Calreticulin using ab92516 at 1/250 dilution in immunohistochemical analysis.

     

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

References

This product has been referenced in:

  • Ou J  et al. ABHD5 blunts the sensitivity of colorectal cancer to fluorouracil via promoting autophagic uracil yield. Nat Commun 10:1078 (2019). Read more (PubMed: 30842415) »
  • Tsalikis J  et al. Isoginkgetin, a Natural Biflavonoid Proteasome Inhibitor, Sensitizes Cancer Cells to Apoptosis via Disruption of Lysosomal Homeostasis and Impaired Protein Clearance. Mol Cell Biol 39:N/A (2019). Read more (PubMed: 30910794) »
See all 21 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Application
Flow Cytometry
Sample
Sacrophilus harrisii (Tasmanian Devil) Cell (C5065 (tasmanian devil tumour cell line))
Permeabilization
Yes - PBS/0.1% tween
Gating Strategy
Population was gated for single cells and DAPI used to exclude dead cells.
Specification
C5065 (tasmanian devil tumour cell line)
Preparation
Cell harvesting/tissue preparation method: Cells were washed in PBS counted and resuspended at 5x10/6 cells per ml in FACS buffer. 100ul was added to tubes for fixation, permeabilisation and staining.
Sample buffer: FACS buffer (PBS/0.5% BSA/0.05% azide)
Fixation
80% methanol

Abcam user community

Verified customer

Submitted Dec 01 2016

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
3% BSA and 0.5% TX100 as blocking agent for 45 minute(s) · Concentration: 3% · Temperature: 25°C
Sample
Monkey Cell (Kidney)
Specification
Kidney
Permeabilization
No
Fixative
3.2% PFA and 0.5% GA at 37C

Dr. Aaron Halpen

Verified customer

Submitted Sep 23 2014

Question
Answer

Thank you for contacting Abcam regarding ab92516. Regarding Flow cytometry, I have confirmed that we used an intracellular cell staining protocol as we reviewed on the phone.  The lab has indicated that the cells need to be fixed with 2% paraformaldehyde. We used permeabilized HeLa cells. The IP protocol is below:   1. Solutions and Reagents 10X Cell Lysis Buffer 200 mM Tris-HCl (pH 7.5) 1.5M NaCl 10 mM Na 2EDTA 10 mM EGTA 10% Triton 25 mM sodium pyrophosphate 10 mM β-glycerophosphate 10 mM Na 3VO 4 (activated) 10 μg/ml leupeptin Add Phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 1mM. (Stock solution 100mM PMSF) NOTE: Add fresh before each use   2.  Immunoprecipitation Protocol  - Mix 200–300 μl of non-denatured cell lysate with appropriate dilutions of primary antibody  - Incubate overnight at 4 °C with gentle rocking.  - Add 25 μl of Anti-Rabbit IgG beads and incubate 2 hours at 4 °C with gentle rocking.  - Microcentrifuge for 30 seconds and discard the supernatant. Wash pellet 3 times with cold PBS.  - Resuspend the pellet with 20 μl of 2x SDS reducing sample buffer containing β-mercaptoethanol (5%, v/v) and heat the sample at 95–100 °C for 3 minutes. - Briefly centrifuge the sample.  - Load 16 μl of sample on to a SDS-PAGE gel or freeze the sample for later use (reboiled for 3 minutes prior loading onto the SDS-PAGE gel). - Perform Western blotting analysis using TrueBlot HRP-conjugated anti-rabbit IgG as secondary antibody We tested HeLa cells. A non-denaturing lysis buffer must be used, since we lyse cells under non-denaturing conditions. I hope this information is helpful.  Please let me know if you have any questions.  

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