Carbonic Anhydrase IX (CA9) Antibody – Mouse Monoclonal

Monoclonal

IgG2a

7H20

Mouse

Supplied as a liquid in saline, 0.5% BSA, 0.09% sodium azide. Labeled with R-Phycoerythrin (PE).

Affinity Purified
Purified by Protein A affinity chromatography from hybridoma culture supernant.

0.025mg/ml (lot specific)

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.

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.

Chromosome: 9; NC_000009.11 (35673915..35681156). Location: 9p12

603179

ModBase 3D Structure for Q16790

Recombinant protein corresponding to Pro59-Asp414 of human Carbonic Anhydrase IX (CA9) expressed in NSO cells.

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 MBS603443, 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.

Antibodies; Abs to Enzymes