Skip to main content

Key Product Details

Validated by

Knockout/Knockdown

Species Reactivity

Human, Mouse, Rat, Bovine, Hamster, Primate

Applications

Block/Neutralize, Dot Blot, Electron Microscopy, Flow Cytometry, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Paraffin, Immunoprecipitation, Knockdown Validated, Protein Array, Simple Western, Western Blot

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free

Concentration

1.0 mg/ml

Product Summary for Calreticulin Antibody - BSA Free

Immunogen

Calreticulin Antibody was developed against a fusion protein to mouse Calreticulin [Uniprot: P14211]

Reactivity Notes

Other species not tested.

Localization

Calreticulin staining is localized to the ER in most cell types and under most conditions.

Marker

Endoplasmic Reticulum Marker

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

48 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for Calreticulin Antibody - BSA Free

Knockdown Validated: Calreticulin Antibody [NB600-101] - Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and Calreticulin knockout (KO) HeLa cell line. PVDF membrane was probed with 1:1500 of Rabbit Anti-Human Calreticulin Polyclonal Antibody (Catalog # NB600-101) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog #HAF008). Specific band was detected for Calreticulin at approximately 55 kDa (as indicated) in the parental HeLa cell line, but is not detectable in the knockout HeLa cell line. This experiment was conducted under reducing conditions.
Western Blot: Calreticulin Antibody [NB600-101] - Human kidney lysate.
Immunocytochemistry/Immunofluorescence: Calreticulin Antibody [NB600-101] - Immunofluorescence staining of Calreticulin in HCT15 colon cancer cells using NB600-101. Secondary antibody was conjugated with Alexa Fluor 488. Photo courtesy of Dr. Birkenkamp Demtroeder, Arhus University Hospital.

Applications for Calreticulin Antibody - BSA Free

Application
Recommended Usage

Flow Cytometry

reported in scientific literature (PMID 19171874)

Immunocytochemistry/ Immunofluorescence

1:50-1:250

Immunohistochemistry

1:50 - 1:200

Immunohistochemistry-Paraffin

1:50 -1:200

Immunoprecipitation

reported in scientific literature (PMID 15161937)

Knockdown Validated

1:1500

Protein Array

reported in scientific literature (PMID 17897946)

Simple Western

1:50

Western Blot

1:1000
Application Notes
In Western blot, a band is observed at ~55 kDa. The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors. In Simple Western only 10 - 15 uL of the recommended dilution is used per data point. Separated by Size-Wes, Sally Sue/Peggy Sue.
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Published Applications

Read 27 publications using NB600-101 in the following applications:

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1.0 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.

Background: Calreticulin

Calreticulin, also called CALR, is an endoplasmic reticulum (ER) protein that functions as a calcium (Ca2+) buffering molecular chaperone (1-3). Together with calnexin and ERp57, calreticulin has a critical role in protein folding and quality control of newly synthesized glycoproteins (1-3). Calreticulin has many diverse functions beyond Ca2+ binding, including cell adhesion, lectin binding, apoptosis, nuclear transport, antigen presentation, and immune response (1-3). Specifically, calreticulin is part of the peptide-loading complex that resides on the ER membrane and is responsible for antigen loading onto MHC class I molecules (1-3). Calreticulin protein has a theoretical molecular weight of 46 kDa and is 417 amino acids (aa) in length (1-3). The protein has three main domains: the N-terminal lectin-like domain, the proline-rich P-domain, and the acidic C-domain which is followed by an ER-retention KDEL signal sequence (1-3).

Given its role in multiple biological processes, it makes sense that calreticulin is implicated in both healthy and disease states. Studies have found that calreticulin mutations were present in patients with myeloproliferative neoplasms (MFN) and essential thrombocythaemia (ET) (4). Calreticulin expression is typically upregulated in most cancer lines, however it is downregulated in some tissues including cervical carcinomas, prostate cancer, and human colon adenocarcinoma (3). High expression of calreticulin on cancer cells is related to tumor cell phagocytosis and is often correlated with, and counteracted by, elevated CD47 expression to prevent cancer cell phagocytosis (3). Calreticulin mutations can serve as a major diagnostic biomarker for MFN and ET and additionally the calreticulin gene may be a potential target for cancer therapeutics (3,4).

References

1. Michalak, M., Groenendyk, J., Szabo, E., Gold, L. I., & Opas, M. (2009). Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum. The Biochemical Journal. https://doi.org/10.1042/BJ20081847

2. Fucikova, J., Spisek, R., Kroemer, G., & Galluzzi, L. (2021). Calreticulin and cancer. Cell Research. https://doi.org/10.1038/s41422-020-0383-9

3. Sun, J., Mu, H., Dai, K., & Yi, L. (2017). Calreticulin: a potential anti-cancer therapeutic target. Die Pharmazie. https://doi.org/10.1691/ph.2017.7031

4. Prins, D., Gonzalez Arias, C., Klampfl, T., Grinfeld, J., & Green, A. R. (2020). Mutant Calreticulin in the Myeloproliferative Neoplasms. HemaSphere. https://doi.org/10.1097/HS9.0000000000000333

Alternate Names

Autoantigen Ro, CALR, Calregulin, cC1qR, CRT, SSA, Vasostatin

Entrez Gene IDs

811 (Human); 12317 (Mouse); 64202 (Rat)

Gene Symbol

CALR

UniProt

Product Documents for Calreticulin Antibody - BSA Free

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot number in the search box below.

Product Specific Notices for Calreticulin Antibody - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Loading...

Protocols

View specific protocols for Calreticulin Antibody - BSA Free (NB600-101):

[[URL:https://www.novusbio.com/products/calreticulin-antibody_nb600-101]][[Ca… Antibody]]
Immunofluorescence Procedure

1) Cell cultures were treated using an HIER [heat-induced epitope retrieval] protocol.
2) Calreticulin polyclonal antibody [NB 600-101] was diluted 1:50 with a DAKO antibody diluent
3) Cultures and primary antibody were incubated at RT for 30 minutes.
4) Cultures were incubated with an Alexa green fluorescent secondary, as per manufacturer'??s protocol.
5) Cultures were observed on a Zeiss Axioconvert microscope and with DigiPix software.

**NOTE: COS7 and HCT29 cell cultures were used as positive controls for this antibody.
[[URL:https://www.novusbio.com/products/calreticulin-antibody_nb600-101]][[Ca… Antibody]]
Immunohistochemistry Procedure

1) Paraffin-embedded sections were treated using an HIER [heat-induced epitope retrieval] protocol.
2) NB 600-101 was diluted 1:50 with a DAKO antibody diluent
3) Sections and primary antibody were incubated at RT for 30 minutes.
4) Sections were incubated with a DAKO secondary, as per manufacturer'??s protocol.
5) Sections were then stained with DAB and H&E.
6) Sections were mounted with Faramount solution.
7) Result of staining was strong.

**NOTE: normal colon mucosa and adenocarcinoma tissues were used as positive controls for this antibody.
[[URL:https://www.novusbio.com/products/calreticulin-antibody_nb600-101]][[Ca… Antibody]]
Western Blot Procedure

1) Scrape cells* off culture dishes and centrifuge.
2) Dissolve cell pellet in decanoyl-N-methyl glucamide (MEGA-10)** and clarify by centrifugation.
3) Mix 30 mg of protein*** with sample buffer containing mercaptoethanol and SDS and run on a 10% SDS gel. The protein was electroblotted on to nitrocellulose.
4) Block nitrocellulose with 5% powdered milk in PBS for 1 hour.
5) Wash the blot with PBS.
6) Add the antibody at a concentration of 1:1000 in 5% powdered milk/PBS and incubate for 1 hour.
7) Wash 3 x 5 minutes with PBS.
8) Add peroxidase-labelled anti-rabbit second antibody in PBS at a concentration of 1:3000 and shake for 1 hour.
9) Wash extensively with PBS.
10) Develop with ECL reagents (Amersham). For this experiment, the film was exposed to the blot for 10 seconds. ****

i. The cells used were the H4IIE rat hepatoma cell line and the NBL-1 bovine renal epithelial cell line.
ii. The detergent used is not critical. MEGA-10 has the advantage of not interfering with the Bradford protein reagent.
iii. Whole cells were used in this experiment. If 30mg of a cell membrane fraction were used a more intense band would be seen.
iv. In this experiment, the antibody was used at 1:1000, but since whole cell protein was used and only 10 seconds development was required it could presumably be used at a lower concentration for many applications.

FAQs for Calreticulin Antibody - BSA Free

Showing  1 - 3 of 3 FAQs Showing All
  • Q: We are looking to buy an anti-calreticulin antibody that would work for WB and IP. Your reference NB 600-101 wouls correspond, but we need it to work on proteins with altered C terminus. Could you please let us know whether the immunizing peptide for this antibody is located N-terminal to AA #364?

    A: Yes, our product NB600-101 was raised against an immunogen that falls between amino acids 150-350 of the mouse Calreticulin protein.

  • Q: I would like to know how the NB600-101 Calreticulin Antibody was used in the neutralization/blocking application. Do you have protocol details for a particular cell type etc?

    A: NB600-101 was validated for B/N via a publication. We do not have a specific protocol available; however, I do have the link to that publication that your customer may find helpful. NCBI site

  • Q: Is this antibody (NB600-101) suitable for immunolabeling in Tokuyasu criosections?

    A: This product has not been tested in Tokuyasu Cryosections but is validated in IHC/ICC/IF. We do assume it should work for you.

Showing  1 - 3 of 3 FAQs Showing All
Loading...