FCM (Flow Cytometry)
(Detection of Carbonic Anhydrase IX/CA9 in U‑87 MG Human Cell Line by Flow Cytometry using AAA14680.)
Mouse Carbonic Anhydrase IX Monoclonal Antibody | anti-CA9 antibody
Carbonic Anhydrase IX (CA9) (PE)
Gene Names
CA9; MN; CAIX
Applications
Flow Cytometry, Functional Assay
Purity
Affinity PurifiedPurified by Protein A affinity chromatography from hybridoma culture supernant.
Synonyms
Carbonic Anhydrase IX, Antibody; Carbonic Anhydrase IX (CA9) (PE); Anti -Carbonic Anhydrase IX (CA9) (PE); anti-CA9 antibody
Host
Mouse
Clonality
Monoclonal
Isotype
IgG2a
Clone Number
7H20
Specificity
Recognizes human Carbonic Anhydrase IX (CA9). Does not crossreact with recombinant mouse CA9 or with recombinant human CA1, 2, 3, 4, 5A, 6, 7, 8, 10, 12, 13, or 14.
Purity/Purification
Affinity Purified
Purified by Protein A affinity chromatography from hybridoma culture supernant.
Purified by Protein A affinity chromatography from hybridoma culture supernant.
Form/Format
Supplied as a liquid in saline, 0.5% BSA, 0.09% sodium azide. Labeled with R-Phycoerythrin (PE).
Concentration
0.025mg/ml (varies by lot)
Applicable Applications for anti-CA9 antibody
Flow Cytometry (FC/FACS)
Application Notes
Suitable for use in Flow Cytometry.
Dilution: Flow Cytometry: Neat. 10ul labels 1x10e6 cells. Tested using U-87 MG human glioblastoma/astrocytoma cell line. Optimal dilutions to be dertermined by the researcher.
Dilution: Flow Cytometry: Neat. 10ul labels 1x10e6 cells. Tested using U-87 MG human glioblastoma/astrocytoma cell line. Optimal dilutions to be dertermined by the researcher.
Immunogen
Recombinant protein corresponding to Pro59-Asp414 of human Carbonic Anhydrase IX (CA9) expressed in NSO cells.
Flow Cytometry Protocol
Reagents Not Provided:
-PBS (Dulbecco's PBS) -BSA
Intended Use:
Designed to quantitatively determine the percentage of cells bearing Carbonic Anhydrase IX (CA9) within a population and qualitatively determine the density of CA9 on cell surfaces by flow cytometry.
Principle of the Test:
Washed cells are incubated with AAA14680, which binds to cells expressing CA9. Unbound phycoerythrin-conjugated antibody is then washed from the cells. Cells expressing CA9 are fluorescently stained: with the intensity of staining directly proportional to the density of expression of CA9. Cell surface expression of CA9 is determined by flow cytometric analysis using 488nm wavelength laser excitation and monitoring emitted fluorescence with a detector optimized to collect peak emissions at 565-605nm.
Sample Preparation:
Peripheral Blood Cells:
Whole blood should be collected in evacuated tubes containing EDTA or heparin as the anticoagulant. Contaminating serum components should be removed by washing the cells three times in an isotonic phosphate buffer (supplemented With 0.50/0 BSA) by centrifugation at 500 x g for 5 minutes. Transfer 5Oul of packed cells to a 5ml tube for staining with the monoclonal antibody. Whole blood will require lysis of RBC following the staining procedure.
Cell Cultures: Continuous cell lines or activated cell cultures should be centrifuged at 500 x g for 5 minutes and washed three times in an isotonic PBS buffer (supplemented with 0.50/0 BSA), as described above, to remove any residual growth factors that may be present in the culture medium. Cells should then be resuspended in the same buffer to a final concentration of 4xl Oe6 cells/ml and 25ul of cells (1 xl Oe5) transferred to a Sml tube for staining.
Note: Adherent cell lines may require pretreatment with O.5mM EDTA to facilitate removal from substrate. Cells that require trypsinization to enable removal from substrate should be further incubated in medium for 6-10 hours on a rocker platform to enable regeneration of the receptors. The use of the rocker platform will prevent reattachment to the substrate.
Sample Staining:
1. Cells should be Fe-blocked by treatment with 1 ug of human IgG/ lOe5 cells for 15 minutes at RT prior to staining. Do not wash excess blocking IgG from this reaction.
2. Transfer 25ul of the Fe-blocked cells (1 xl Oe5 cells) or SOul of packed whole blood to a 5ml tube.
3. Add 1 Oul of Cll 05-80A. Incubate for 30-45 minutes at 4°C.
4. Following this incubation, remove unreacted CA9 reagent by washing the cells twice in 4ml of the same PBS buffer (note: whole blood will require an RBC lysis step at this point using any commercially available lysing reagent).
5. Finally, resuspend the cells in 200-400ul of PBS buffer for final flow cytometric analysis.
6. As a control for analysis, cells in a separate tube should be treated with PE-Iabeled mouse IgG2a antibody.
Optimai conditions should be determined by each laboratory.
-PBS (Dulbecco's PBS) -BSA
Intended Use:
Designed to quantitatively determine the percentage of cells bearing Carbonic Anhydrase IX (CA9) within a population and qualitatively determine the density of CA9 on cell surfaces by flow cytometry.
Principle of the Test:
Washed cells are incubated with AAA14680, which binds to cells expressing CA9. Unbound phycoerythrin-conjugated antibody is then washed from the cells. Cells expressing CA9 are fluorescently stained: with the intensity of staining directly proportional to the density of expression of CA9. Cell surface expression of CA9 is determined by flow cytometric analysis using 488nm wavelength laser excitation and monitoring emitted fluorescence with a detector optimized to collect peak emissions at 565-605nm.
Sample Preparation:
Peripheral Blood Cells:
Whole blood should be collected in evacuated tubes containing EDTA or heparin as the anticoagulant. Contaminating serum components should be removed by washing the cells three times in an isotonic phosphate buffer (supplemented With 0.50/0 BSA) by centrifugation at 500 x g for 5 minutes. Transfer 5Oul of packed cells to a 5ml tube for staining with the monoclonal antibody. Whole blood will require lysis of RBC following the staining procedure.
Cell Cultures: Continuous cell lines or activated cell cultures should be centrifuged at 500 x g for 5 minutes and washed three times in an isotonic PBS buffer (supplemented with 0.50/0 BSA), as described above, to remove any residual growth factors that may be present in the culture medium. Cells should then be resuspended in the same buffer to a final concentration of 4xl Oe6 cells/ml and 25ul of cells (1 xl Oe5) transferred to a Sml tube for staining.
Note: Adherent cell lines may require pretreatment with O.5mM EDTA to facilitate removal from substrate. Cells that require trypsinization to enable removal from substrate should be further incubated in medium for 6-10 hours on a rocker platform to enable regeneration of the receptors. The use of the rocker platform will prevent reattachment to the substrate.
Sample Staining:
1. Cells should be Fe-blocked by treatment with 1 ug of human IgG/ lOe5 cells for 15 minutes at RT prior to staining. Do not wash excess blocking IgG from this reaction.
2. Transfer 25ul of the Fe-blocked cells (1 xl Oe5 cells) or SOul of packed whole blood to a 5ml tube.
3. Add 1 Oul of Cll 05-80A. Incubate for 30-45 minutes at 4°C.
4. Following this incubation, remove unreacted CA9 reagent by washing the cells twice in 4ml of the same PBS buffer (note: whole blood will require an RBC lysis step at this point using any commercially available lysing reagent).
5. Finally, resuspend the cells in 200-400ul of PBS buffer for final flow cytometric analysis.
6. As a control for analysis, cells in a separate tube should be treated with PE-Iabeled mouse IgG2a antibody.
Optimai conditions should be determined by each laboratory.
Preparation and Storage
May be stored at 4°C in the dark. DO NOT FREEZE! Stable for 12 months at 4°C after received as an undiluted liquid. Further dilutions can be made in assay buffer.
Caution: PE conjugates are sensitive to light. Centrifuge before opening to ensure complete recovery of vial contents.
Caution: PE conjugates are sensitive to light. Centrifuge before opening to ensure complete recovery of vial contents.
FCM (Flow Cytometry)
(Detection of Carbonic Anhydrase IX/CA9 in U‑87 MG Human Cell Line by Flow Cytometry using AAA14680.)
FCM (Flow Cytometry)
(Detection of Carbonic Anhydrase IX/CA9 in U‑87 MG Human Cell Line by Flow Cytometry using AAA14680.)
Related Product Information for anti-CA9 antibody
Carbonic anhydrase (CA) catalyzes the reversible reaction of CO2 + H2O = HCO3- + H+, which is fundamental to many processes such as respiration, renal tubular acidification and bone resorption. CA9, also known as membrane antigen MN and renal cell carcinoma (RCC)-associated antigen G250, is a transmembrane enzyme expressed primarily in carcinoma cells. It is one of the best markers for hypoxia and for RCC.
Product Categories/Family for anti-CA9 antibody
NCBI and Uniprot Product Information
NCBI GI #
NCBI GeneID
NCBI Official Full Name
carbonic anhydrase IX
NCBI Official Synonym Full Names
carbonic anhydrase IX
NCBI Official Symbol
CA9
NCBI Official Synonym Symbols
MN; CAIX
NCBI Protein Information
carbonic anhydrase 9; pMW1; CA-IX; P54/58N; OTTHUMP00000022773; membrane antigen MN; carbonic dehydratase; carbonate dehydratase IX; RCC-associated antigen G250; RCC-associated protein G250; renal cell carcinoma-associated antigen G250
UniProt Protein Name
Carbonic anhydrase 9
UniProt Gene Name
CA9
UniProt Synonym Gene Names
G250; MN
UniProt Entry Name
CAH9_HUMAN
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Product Notes
The CA9 ca9 (Catalog #AAA14680) is an Antibody produced from Mouse and is intended for research purposes only. The product is available for immediate purchase. AAA Biotech's Carbonic Anhydrase IX can be used in a range of immunoassay formats including, but not limited to, Flow Cytometry (FC/FACS). Suitable for use in Flow Cytometry. Dilution: Flow Cytometry: Neat. 10ul labels 1x10e6 cells. Tested using U-87 MG human glioblastoma/astrocytoma cell line. Optimal dilutions to be dertermined by the researcher. Researchers should empirically determine the suitability of the CA9 ca9 for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process. It is sometimes possible for the material contained within the vial of "Carbonic Anhydrase IX, Monoclonal Antibody" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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