Carbonic Anhydrase IX/CA9 Antibody (2D3)

Western Blot: Carbonic Anhydrase IX/CA9 Antibody (2D3) [NBP1-51691] - Carbonic Anhydrase IX mouse antibody against Hela (1) and A549 (2) cell lysates. Bands were detected at a molecular weight of approximately 50 kDa in both cell lines.

Immunohistochemistry-Paraffin: Carbonic Anhydrase IX/CA9 Antibody (2D3) [NBP1-51691] - Paraffin-embedded lung tissues (left) and colonic tissues (right) using Carbonic Anhydrase IX mouse antibody with DAB staining.

Immunocytochemistry/Immunofluorescence: Carbonic Anhydrase IX/CA9 Antibody (2D3) [NBP1-51691] - Mouse choroid stained with anti-Carbonic Anhydrase IX antibody. Image from verified customer review.

Flow Cytometry: Carbonic Anhydrase IX/CA9 Antibody (2D3) [NBP1-51691] - A surface stain was performed on A431 cells with Carbonic Anhydrase IX/CA9 NBP1-51691 (blue) and a matched isotype control NBP2-27287 (orange). Cells were incubated in an antibody dilution of 1 ug/mL for 20 minutes at room temperature, followed by Dylight488-conjugated anti-mouse secondary antibody.

Immunocytochemistry/Immunofluorescence: Carbonic Anhydrase IX/CA9 Antibody (2D3) [NBP1-51691] - HeLa cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton X-100. The cells were incubated with anti-CA9 (2D3) [NBP1-51691] at a 1:200 dilution overnight at 4C and detected with an anti-mouse Dylight 488 (Green) at a 1:500 dilution. Actin was detected with Phalloidin 568 (Red) at a 1:200 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.

Flow Cytometry: Carbonic Anhydrase IX/CA9 Antibody (2D3) [NBP1-51691] - Analysis of NTERA-2 cells using Carbonic Anhydrase IX mouse mAb (green) and negative control (purple).

• Reactivity: Hu
• Applications: WB, ELISA, Flow, Flow-IC, ICC/IF, IHC, IHC-P, CyTOF-ready
• Clone: 2D3
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated
• Concentration: 1.0 mg/ml

Carbonic Anhydrase IX/CA9 Antibody (2D3) Summary

• Immunogen: This Carbonic Anhydrase IX/CA9 Antibody (2D3) was made to a purified recombinant fragment of human Carbonic Anhydrase IX expressed in E. coli [UniProt# Q16790].
• Marker: Hypoxia Marker
• Isotype: IgG1
• Clonality: Monoclonal
• Host: Mouse
• Gene: CA9
• Purity: Protein A or G purified

Applications/Dilutions

• Dilutions:
  • Western Blot 1:2000
  • ELISA 1:10000
  • Flow Cytometry 1:200 - 1:400
  • Immunocytochemistry/Immunofluorescence
  • Immunohistochemistry 1:10 - 1:500
  • Immunohistochemistry-Paraffin 1:200 - 1:1000
  • CyTOF-ready
  • Flow (Intracellular) 1 ug/mL
• Application Notes: 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.
• Theoretical MW: 50 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:

  HeLa Whole Cell Lysate

Packaging, Storage & Formulations

• Storage: Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
• Buffer: PBS
• Preservative: 0.05% Sodium Azide
• Concentration: 1.0 mg/ml
• Purity: Protein A or G purified

Alternate Names for Carbonic Anhydrase IX/CA9 Antibody (2D3)

• CA9
• CAIX
• CA-IX
• Carbonate dehydratase IX
• carbonic anhydrase 9
• Carbonic Anhydrase IX
• carbonic dehydratase
• EC 4.2.1.1
• G250
• Membrane antigen MN
• MN
• P54/58N
• PMW1
• RCC
• RCC-associated antigen G250
• RCC-associated protein G250
• Renal cell carcinoma-associated antigen G250

Background

Carbonic anhydrase alpha, isozyme IX, belongs to a family of zinc-containing metalloproteins which hydrate carbon dioxide to generate bicarbonate ions and protons (1). This main catalytic function allows carbonic anhydrase IX to participate in cellular pH regulation. The large family of carbonic anhydrase metalloproteins includes three major classes which have been identified based on sequence and structure analysis. The alpha class is a monomer found in mammals. The beta class may occur as a dimer, tetramer, hexamer or octamer and is found in plants, algae, and bacteria. Lastly, the gamma class is a trimer found in bacteria and represents the most ancient carbonic anhydrase. These three classes of carbonic anhydrase enzymes lack sequence or structural similarities, but all share a conserved active site zinc atom (1).

Carbonic anhydrase IX (theoretical molecular weight 50kDa) belongs to the monomeric alpha class and is a single pass-transmembrane protein with two extracellular domains which serve catalytic and cell adhesion functions (2, 3). By cooperating with sodium bicarbonate cotransporters (NBC), lactate and proton exporting monocarboxylic acid transporters (MCT), and a sodium/hydrogen exchanger (NHE), carbonic anhydrase IX is involved in pH regulation across the cell membrane. This functional property protects cancer cells from intracellular acidification and partly explains the role of carbonic anhydrase IX in cancer cell survival and proliferation. In contrast, the pH regulating activity of carbonic anhydrase IX induces extracellular acidification, which has been implicated in epithelial to mesenchymal transition (EMT) and promoting cancer invasion. Carbonic anhydrase IX is frequently overexpressed in cancer cells (e.g., colorectal-, breast-, lung-carcinoma and brain tumors), an effect promoted by hypoxia within the tumor microenvironment (4). An exception are tumors carrying pVHL inactivating mutations, such as clear cell renal cell carcinoma (ccRCC), where HIF-alpha is stabilized due to dysfunctional proteasomal targeting and can induce HRE (Hypoxia Response Element) containing genes even under physiological normoxia (5). Carbonic anhydrase IX may be detected by immunostaining in tumors, which is found in association with necrotic tissue and metastatic cells. Because the expression of carbonic anhydrase IX correlates with both tumor grade and stage, analysis of its expression in tumors serves as a prognostic factor (4, 6).

References
1. Tripp, B. C., Smith, K., & Ferry, J. G. (2001). Carbonic Anhydrase: New Insights for an Ancient Enzyme. Journal of Biological Chemistry. https://doi.org/10.1074/jbc.R100045200

2. Nishimori, I., & Onishi, S. (2001). Carbonic anhydrase isozymes in the human pancreas. Digestive and Liver Disease. https://doi.org/10.1016/s1590-8658(01)80138-9

3. Zavadova, Z., & Zavada, J. (2005). Carbonic anhydrase IX (CA IX) mediates tumor cell interactions with microenvironment. Oncology Reports. https://doi.org/10.3892/or.13.5.977

4. Pastorekova, S., & Gillies, R. J. (2019). The role of carbonic anhydrase IX in cancer development: links to hypoxia, acidosis, and beyond. Cancer and Metastasis Reviews. https://doi.org/10.1007/s10555-019-09799-0

5. Haase, V. (2009). The VHL Tumor Suppressor: Master Regulator of HIF. Current Pharmaceutical Design. https://doi.org/10.2174/138161209789649394

6. Young, J. R., Coy, H., Kim, H. J., Douek, M., Sisk, A., Pantuck, A. J., & Raman, S. S. (2018). Association of the gross appearance of intratumoral vascularity at MDCT with the carbonic anhydrase IX score in clear cell renal cell carcinoma. American Journal of Roentgenology. https://doi.org/10.2214/AJR.18.19725

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 Carbonic Anhydrase IX/CA9 Antibody (NBP1-51691)(5)

Publications using NBP1-51691

• Li Z, Jiang L, Chew SH et al. Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia Redox Biol Aug 10 2019 [PMID: 31442913] (WB, Human)
• Kelly NJ, Varga JFA, Specker EJ, Romeo CM. Hypoxia activates cadherin-22 synthesis via eIF4E2 to drive cancer cell migration, invasion and adhesion. Oncogene. 2018 Feb 01 [PMID: 28991229] (WB, Human)
• Federici C, Lugini L, Marino ML et al. Lansoprazole and carbonic anhydrase IX inhibitors sinergize against human melanoma cells. J Enzyme Inhib Med Chem. May 3 2016 [PMID: 27142956] (WB, Human)
• Cai C, Alshehri A, Choksi R, Pestronk A. Regional ischemic immune myopathy: a paraneoplastic dermatomyopathy. J. neuropathol. Exp. neurol. 2014 Dec 01 [PMID: 25383636] (IHC, Human)
• Persano L, Pistollato F, Rampazzo E et al. BMP2 sensitizes glioblastoma stem-like cells to Temozolomide by affecting HIF-1 alpha stability and MGMT expression Cell Death Dis 2012 Oct 18 [PMID: 23076220] (WB, Human)

Review for Carbonic Anhydrase IX/CA9 Antibody (NBP1-51691) (1)

Average Rating: 5

(Based on 1 review)

We have 1 review tested in 1 species: Human.

reviewed by: Anonymous WB Human 11/20/2015

Summary

• Application: Western Blot
• Sample Tested: MCF-7 whole cell lysate
• Species: Human
• Lot: A2

Blocking

• Blocking Details: PBST with 5% non-fat dry milk, 1 h, Room Temperature

Primary Anitbody

• Dilution Ratio: 1:1000

Secondary Antibody

• Secondary Description: Goat anti-mouse IgG HRP
• Secondary Manufacturer Cat#: sc-2005
• Secondary Concentration: 1:2000

Details

• Detection Notes: Chemiluminescence, GBox Chemi XL1.4 from Syngene, 12 sec, no positive or negative controls

Comments

• Comments: This antibody can be used at much higher dilution. At lower dilution (less than 1:1000) or if high amount of protein is loaded multiple bands may be seen.

FAQs for Carbonic Anhydrase IX/CA9 Antibody (NBP1-51691). (Showing 1 - 3 of 3 FAQs).

1. We are looking for the antibody available for cell sorting by CA9. The sorted cells must be cultured for the expansion after the sorting for the following experiments. Do you have the antibodies useful for this?
   • We sell two carbonic anhydrase IX antibodies that have been validated for Flow Cytometry detection of the human protein. These have catalogue numbers NBP1-51691 and NBP1-47688 and can be seen at the following link: carbonic anhydrase IX antibodies. Since both of these products contain sodium azide as a preservative, you are unlikely to be able to culture the cells on following sorting unless you performed a buffer exchange prior to using the antibody. Unfortunately we have no direct experience with either of these products of staining and subsequently expanding cell populations.
2. I am looking for a primary antibody to Carbonic Anhydrase 9 and I want an antibody that recognises an extracellular epitope so that I can use it for live cell flow cytometry. Would #NBP1-51691 be a suitable candidate?
   • The immunogen of our Carbonic Anhydrase IX antibody (clone 2D3) # NBP1-51691 corresponds to peptide from amino acids region between 40-290 of human CA9, which appears to be in the extracellular domain of this protein (based on UniProt: Q16790). In our QC lab, this antibody has been validated for FLOW application, so that you may use this antibody for your current experiments.
3. What is the predicted theoretical molecular weight of Carbonic Anhydrase IX/CA9 antibodies?
   • The TMW for Carbonic Anhydrase IX/CA9 antibodies is 50 kDa. Please 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.

Bioinformatics

• Gene Symbol: CA9
• Entrez:
  • 768(Human)
• Uniprot:
  • Q16790()