Calreticulin Antibody - BSA Free

Western Blot: 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.

Immunohistochemistry-Paraffin: Calreticulin Antibody [NB600-101] - Analysis of a FFPE tissue section of human thyroid gland using 1:50 dilution of Calreticulin antibody. The signal was developed using HRP-DAB based detection method which followed counterstaining of the nuclei with hematoxylin. This representative section shows a strong positivity of Calreticulin in the follicular epithelial cells, wherein the signal was found to be very intense in the perinuclear region of the cells which correlates well with Endoplasmic reticulum localization of this protein. The para-follicular cells, endothelial cells of blood vessels (not the RBCs though) and the loose connective tissue in the section showed a weak cytoplasmic staining. Some staining was observed in the follicles/colloids also which is potentially the secreted form of Calreticulin.

Immunocytochemistry/Immunofluorescence: Calreticulin Antibody [NB600-101] - The Calreticulin antibody NB600-101 was tested in HeLa cells at a 1:250 dilution against DyLight 488 (Green). Alpha-tubulin and nuclei were counterstained against DyLight 550 (Red) and DAPI (Blue), respectively.

Immunohistochemistry-Paraffin: Calreticulin Antibody [NB600-101] - Analysis of a FFPE tissue section of human thyroid gland using 1:50 dilution of Calreticulin antibody. The signal was developed using HRP-DAB based detection method which followed counterstaining of the nuclei with hematoxylin. This representative section shows a strong positivity of Calreticulin in the follicular epithelial cells, wherein the signal was found to be very intense in the perinuclear region of the cells which correlates well with Endoplasmic reticulum localization of this protein. The para-follicular cells, endothelial cells and the loose connective tissue in the section showed a weak cytoplasmic staining. Some staining was observed in the follicles/colloids also which is potentially the secreted form of Calreticulin.

Simple Western: Calreticulin Antibody [NB600-101] - Lane view shows a specific band for Calreticulin in 0.5 mg/ml of HeLa lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.

Calreticulin was detected in immersion fixed U-2 OS human osteosarcoma cell line using Rabbit anti-Calreticulin Affinity Purified Polyclonal Antibody conjugated to Alexa Fluor® 647 (Catalog # NB600-101AF647) (light blue) at 10 µg/mL overnight at 4C. Cells were counterstained with DAPI (blue). Cells were imaged using a 40X objective.

• Reactivity: Hu, Mu, Rt, Bv, Ha, Pm
• Applications: WB, Simple Western, DB, Flow, ICC/IF, IHC, IP, MA, MS, B/N, KD
• Clonality: Polyclonal
• Host: Rabbit
• Conjugate: Unconjugated
• Format: BSA Free
• Concentration: 1.0 mg/ml

Calreticulin Antibody - BSA Free Summary

• Description: Novus Biologicals Rabbit Calreticulin Antibody - BSA Free (NB600-101) is a polyclonal antibody validated for use in IHC, WB, Flow, ICC/IF, Simple Western and IP. Anti-Calreticulin Antibody: Cited in 28 publications. All Novus Biologicals antibodies are covered by our 100% guarantee.
• Immunogen: Calreticulin Antibody was developed against a fusion protein to mouse Calreticulin [Uniprot: P14211]
• Localization: Calreticulin staining is localized to the ER in most cell types and under most conditions.
• Marker: Endoplasmic Reticulum Marker
• Isotype: IgG
• Clonality: Polyclonal
• Host: Rabbit
• Gene: CALR
• Purity: Immunogen affinity purified

Applications/Dilutions

• Dilutions:
  • Block/Neutralize
  • Dot Blot
  • Electron Microscopy
  • 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.
  See Simple Western Antibody Database for Simple Western validation: Tested in HeLa lysate 0.5 mg/mL, separated by Size, antibody dilution of 1:50, apparent MW was 64 kDa. Separated by Size-Wes, Sally Sue/Peggy Sue.
• 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.
• Control:

  Calreticulin Overexpression Lysate

Reactivity Notes

Other species not tested.

Packaging, Storage & Formulations

• Storage: Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.
• Buffer: PBS
• Preservative: 0.02% Sodium Azide
• Concentration: 1.0 mg/ml
• Purity: Immunogen affinity purified

Alternate Names for Calreticulin Antibody - BSA Free

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

Background

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

Limitations

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.

Publications for Calreticulin Antibody (NB600-101)(29)

Publications using NB600-101

• Sanchez-Herrero E, Campos-Silva C, Caceres-Martell Y et al. ALK-Fusion Transcripts Can Be Detected in Extracellular Vesicles (EVs) from Nonsmall Cell Lung Cancer Cell Lines and Patient Plasma: Toward EV-Based Noninvasive Testing Clinical chemistry [PMID: 35348673] (ICC/IF, Human)
• Zheng C, Sui B, Zhang X et al. Apoptotic vesicles restore liver macrophage homeostasis to counteract type 2 diabetes Journal of Extracellular Vesicles 2021-05-01 [PMID: 34084287] (Human)
• Harada K, Matsuoka H, Toyohira Y et al. Mechanisms for establishment of GABA signaling in adrenal medullary chromaffin cells Journal of neurochemistry 2021-03-11 [PMID: 33704788]
• Roberti MP, Yonekura S, Duong CPM et al. Chemotherapy-induced ileal crypt apoptosis and the ileal microbiome shape immunosurveillance and prognosis of proximal colon cancer Nat. Med. 2020-05-25 [PMID: 32451498] (Mouse)
• Musante L, Bontha Sv, La Salvia S et Al. Rigorous characterization of urinary extracellular vesicles (uEVs) in the low centrifugation pellet - a neglected source for uEVs Sci Rep 2020-02-28 [PMID: 32111925] (Western Blot, Immunocytochemistry/ Immunofluorescence, Mouse)
  
  Details:
  Antibody was used in Western Blot analysis to verify that subcellular contaminants were not interfering with detecting target bio-markers.
• Musante L, Bontha Sv, La Salvia S et Al. Rigorous characterization of urinary extracellular vesicles (uEVs) in the low centrifugation pellet - a neglected source for uEVs Sci Rep Feb 28 2020 [PMID: 32111925] (WB, Human)
• Lekszas C, Foresti O, Raote I et Al. Biallelic TANGO1 mutations cause a novel syndromal disease due to hampered cellular collagen secretion Elife 2020-02-26 [PMID: 32101163] (ICC/IF, Human)
• Sikorski K, Mehta A et al. A high-throughput pipeline for validation of antibodies. Nat Methods 2018-01-11 [PMID: 30377371] (Human)
  
  Details:
  Antibody validation based on denaturing gel electrophoresis of biotinylated cell lysates (PAGE) followed by mass spectrometry (MS) and antibody array analysis (MAP).
• Dong X, Wu W, Ma L et al. Collectin-11 Is an Important Modulator of Retinal Pigment Epithelial Cell Phagocytosis and Cytokine Production J Innate Immun 2017-08-04 [PMID: 28772263] (FLOW, Mouse)
• Yamada HY, Kumar G, Zhang Y et al. Systemic chromosome instability in Shugoshin-1 mice resulted in compromised glutathione pathway, activation of Wnt signaling and defects in immune system in the lung. Oncogenesis. 2016-08-16 [PMID: 27526110] (WB, IF/IHC)
• Teng CY, van Oers MM, Wu TY. Additive effect of calreticulin and translation initiation factor eIF4E on secreted protein production in the baculovirus expression system. Appl Microbiol Biotechnol. 2013-10-01 [PMID: 23900798] (WB)
• Teng CY, Chang SL, Tsai MF, Wu TY. alpha-Synuclein and beta-synuclein enhance secretion protein production in baculovirus expression vector system Appl Microbiol Biotechnol 2013-01-12 [PMID: 23314197] (WB)
• Teng CY, Chang SL, van Oers MM, Wu TY. Enhanced Protein Secretion From Insect Cells by Co-Expression of the Chaperone Calreticulin and Translation Initiation Factor eIF4E. Mol Biotechnol. 2012-05-04 [PMID: 22555850] (WB)
• Bazwinsky-Wutschke I, Wolgast S, Muhlbauer E, Peschke E. Distribution patterns of calcium-binding proteins in pancreatic tissue of non-diabetic as well as type 2 diabetic rats and in rat insulinoma beta-cells (INS-1). Histochem Cell Biol. 2010-07-07 [PMID: 20607274] (Rat)
• erson DJ, Vargas JD, Hsiao JP et al. Recruitment of functionally distinct membrane proteins to chromatin mediates nuclear envelope formation in vivo. J Cell Biol;186(2):183-191. 2009-01-01 [PMID: 19620630] (WB, ICC/IF)
• Baruah P et al. C1q enhances IFN-gamma production by antigen-specific T cells via the CD40 costimulatory pathway on dendritic cells. Blood113(15):3485-93. 2009-04-09 [PMID: 19171874] (FLOW, ICC/IF, Mouse)
• Jain, P et al. Identification of Human T Cell Leukemia Virus Type 1 Tax Amino Acid Signals Cellular Factors Involved in Secretion of the Viral Oncoprotein. J Biol Chem; 282:34581-34593. 2007-11-01 [PMID: 17897946] (PA, Cytometric Bead Assay Standard, Hamster)
• Zhou M, He HJ, Suzuki R et al. Localization of sulfonylurea receptor subunits, SUR2A and SUR2B, in rat heart. J Histochem Cytochem 2007-08-01 [PMID: 17438353] (Rat)
• Nurden P et al. Impaired megakaryocytopoiesis in type 2B von Willebrand disease with severe thrombocytopenia. Blood108(8):2587-95. 2006-10-15 [PMID: 16720832] (WB, Human)
• Zhou M et al. ATP-sensitive K+-channel subunits on the mitochondria and endoplasmic reticulum of rat cardiomyocytes. J Histochem Cytochem53(12):1491-500. 2005-12-01 [PMID: 15983113] (IF/IHC, WB, Rat)
• Tsuzaka K, Setoyama Y, Yoshimoto K et al. A splice variant of the TCR zeta mRNA lacking exon 7 leads to the down-regulation of TCR zeta, the TCR/CD3 complex, and IL-2 production in systemic lupus erythematosus T cells. J Immunol174(6):3518-25. 2005-03-15 [PMID: 15749888] (ICC/IF, Human)
• Olesen SH et al. Human FK506 binding protein 65 is associated with colorectal cancer. Mol Cell Proteomics4(4):534-44. 2005-04-01 [PMID: 15671042] (ICC/IF, WB, IF/IHC, Human)
• Khanna R, Lee EJ, Papazian DM. Transient calnexin interaction confers long-term stability on folded K+ channel protein in the ER. J Cell Sci117(Pt 14):2897-908. 2004-06-15 [PMID: 15161937] (IP, Human)
• Knox CD et al. Novel role of phospholipase C-delta1: regulation of liver mitochondrial Ca2+ uptake. Am J Physiol Gastrointest Liver Physiol287(3):G533-40. 2004-09-01 [PMID: 15107298] (WB, Rat)
• Somogyi E et al. Calreticulin--an endoplasmic reticulum protein with calcium-binding activity is also found in the extracellular matrix. Matrix Biol22(2):179-91. 2003-04-01 [PMID: 12782144] (EM, Rat)
• Ho HC, Suarez SS. Characterization of the intracellular calcium store at the base of the sperm flagellum that regulates hyperactivated motility. Biol Reprod68(5):1590-6. 2003-05-01 [PMID: 12606347] (ICC/IF, Bovine)
• Luo X et al. C1q-calreticulin induced oxidative neurotoxicity: relevance for the neuropathogenesis of Alzheimer's disease. J Neuroimmunol135(1-2):62-71. 2003-02-01 [PMID: 12576225] (B/N, ICC/IF, Rat)
• Placantonakis D, Cicirata F, Welsh JP. A dominant negative mutation of neuronal connexin 36 that blocks intercellular permeability. Brain Res Mol Brain Res98(1-2):15-28. 2002-01-31 [PMID: 11834292] (ICC/IF, Primate)
• Woods AJ et al. Paxillin associates with poly(A)-binding protein 1 at the dense endoplasmic reticulum and the leading edge of migrating cells. J Biol Chem277(8):6428-37. 2002-02-22 [PMID: 11704675] (ICC/IF, WB, Mouse)

FAQs for Calreticulin Antibody (NB600-101). (Showing 1 - 3 of 3 FAQs).

1. 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?
   • Yes, our product NB600-101 was raised against an immunogen that falls between amino acids 150-350 of the mouse Calreticulin protein.
2. 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?
   • 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
3. Is this antibody (NB600-101) suitable for immunolabeling in Tokuyasu criosections?
   • This product has not been tested in Tokuyasu Cryosections but is validated in IHC/ICC/IF. We do assume it should work for you.

Bioinformatics

• Gene Symbol: CALR
• Entrez:
  • 811(Human)
  • 12317(Mouse)
  • 64202(Rat)
• Uniprot:
  • P14211(Mouse)