Overview

  • Product name
    Anti-Calreticulin antibody [FMC 75]
    See all Calreticulin primary antibodies
  • Description
    Mouse monoclonal [FMC 75] to Calreticulin
  • Host species
    Mouse
  • Tested applications
    Suitable for: WB, IP, Flow Cyt, ICC/IF, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Monkey
    Predicted to work with: Chinese hamster
  • Immunogen

    Fusion protein corresponding to Calreticulin. Calreticulin-maltose binding fusion protein.

  • Positive control
    • WB: HeLa whole cell lysate. IHC-P: Human placenta tissue. ICC/IF: HeLa cell lysate. Flow: HeLa cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab22683 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. Predicted molecular weight: 63 kDa.
IP Use a concentration of 12.5 µg/ml.
Flow Cyt Use 1-2µg for 106 cells.

(Additional resource: PMID - 18689689)

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

ICC/IF 1/200.

Fix and permeabilize cells prior to staining.

IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

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

  • ICC/IF image of ab22683 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab22683, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Ab22683 staining human placenta. Staining is localised to the cytoplasm.
    Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control.
    Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer citrate pH 6.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS), then incubated with primary antibody for 20 minutes, and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
  • Anti-Calreticulin antibody [FMC 75] (ab22683) at 1/2000 dilution + HEK293 whole cell lysate at 10 µg

    Secondary
    HRP-conjugated goat anti-mouse IgG (H+L) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 63 kDa
    Observed band size: 42,65 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 30 kDa (possible non-specific binding)


    Exposure time: 10 seconds


    Blocked with 5% milk for 1 hour at 23°C.

    Incubated with the primary antibody in PBS + 0.5% Tween 20 for 12 hours at 4°C.

    See Abreview

  • Overlay histogram showing HeLa cells stained with ab22683 (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 (ab22683, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. 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.

References

This product has been referenced in:
  • Zhao D  et al. Cardiac-derived CTRP9 protects against myocardial ischemia/reperfusion injury via calreticulin-dependent inhibition of apoptosis. Cell Death Dis 9:723 (2018). Read more (PubMed: 29925877) »
  • Tao Y  et al. Correlation of five secretory proteins with the nasopharyngeal carcinoma metastasis and the clinical applications. Oncotarget 8:29383-29394 (2017). Read more (PubMed: 28107202) »
See all 36 Publications for this product

Customer reviews and Q&As

1-10 of 33 Abreviews or Q&A

Application
Immunocytochemistry
Sample
Human Cultured Cells (OV90 cells)
Permeabilization
Yes - 0.1% TritonX-100
Specification
OV90 cells
Blocking step
BSA as blocking agent for 18 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 20°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jun 22 2018

Application
Western blot
Sample
Mouse Cell lysate - whole cell (Liver Extract)
Gel Running Conditions
Reduced Denaturing (4-20% Tris Glycin)
Loading amount
20 µg
Specification
Liver Extract
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Oct 25 2017

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (B16 tumors taken out of C57bl mice)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate pH 6
Permeabilization
No
Specification
B16 tumors taken out of C57bl mice
Blocking step
Innovex Background Buster as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 20°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Sep 19 2017

Application
Western blot
Sample
Human Cell lysate - whole cell (OV90 cells)
Gel Running Conditions
Reduced Denaturing (4-20% Tris Glycin gel)
Loading amount
800000 cells
Specification
OV90 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 Aug 19 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (B16 cell line)
Permeabilization
Yes - 0.25% Triton X-100
Specification
B16 cell line
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 20°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Aug 12 2016

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (N87 cancer cell line)
Permeabilization
Yes - 0.25% Triton X-100
Specification
N87 cancer cell line
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 20°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jun 22 2016

Answer

According to http://www.uniprot.org/uniprot/P27797 Calreticulin is localized to the ER, cytoplasm, secreted, and on the cell surface, so rather than being a multipass membrane protein it is translocated to different regions of the cell. Thus, uniprot does not list specific amino acid ranges of the protein that are intracellular vs. extracellular when it is localized on the cell surface, and since it is most prevalent in the ER and cytoplasm our anti-Calreticulin antibodies were validated in ICC and flow with fixed, permeabilized cells. Thus, I am not sure whether or not our anti-Calreticulin antibodies, including ab22683, will bind to Calreticulin on the cell surface because I do not know what portion of the protein is extracellular vs. intracellular when it is surface-localized.

Read More

Answer

ab22683's immunogen was Calreticulin-maltose binding fusion protein. I am very sorry but we have not done epitope mapping for this antibody and therefore cannot confirm exactly where it binds so unfortunately I cannot say whether it will recognise your mutant protein.

For ab16144, we do not have the exact sequence used to make this antibody. However, the antibody was made to recombinant protein. So it's possible that it will work with their mutant protein but we cannot guarantee this. Since this antibody is a polyclonal antibody, determining the exact epitope would be difficult at best when using a recombinant protein antigen.

Read More

Answer

We have a flow-image with Hela cells on the online datasheet. The protocol for this is as follows:

Overlay histogram showing HeLa cells stained with ab22683 (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 (ab22683, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (https://www.abcam.com/ab96879.html) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (https://www.abcam.com/ab91353.html, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. 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.


We are also aware that a customer published flow data with this antibody:

Park YJ et al. PAI-1 inhibits neutrophil efferocytosis. Proc Natl Acad Sci U S A 105:11784-9 (2008). ICC/IF, Flow Cyt ; Mouse . PubMed: 18689689

The protocol for this one would be in the publication, or you might need to contact the researchers for more detailed information.

Read More

Answer

Thank you for contacting us.

I can confirm that Anti-Calreticulin antibody [FMC 75] (ab22683) has been tested, and it is therefore guaranteed to work in Flow Cytometry with the species mentioned on the datasheet.

All of our products are covered by our Abpromise® guarantee, which ensures that you can trust our products, and they should work in the tested species and applications stated on the datasheet, or we will offer a replacement, credit, or refund, if reported within 6 months of purchase.

I hope this information is helpful. Please do not hesitate to contact us if you need further advice or information.

Read More

1-10 of 33 Abreviews or Q&A

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