Kit Rat Monoclonal Antibody [Clone ID: ACK4]

CAT#: CL042B

Kit rat monoclonal antibody, clone ACK4, Biotin

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Specifications

Product Data

• Clone Name: ACK4
• Applications: FC
• Recommended Dilution: Flow Cytometry.
• Reactivities: Mouse
• Host: Rat
• Isotype: IgG2a
• Clonality: Monoclonal
• Immunogen: IL-3 dependent mast cells derived from WB- +/+ mice
  Donor: Wistar spleen
  Fusion Partner: X63.653. Ag8
• Specificity: Anti-mouse CD117 monoclonal antibody recognizes the receptor tyrosine kinase, c-kit. The ligand for this receptor is steel factor (stem cell factor), which exists in both soluble and membrane form. The interaction between steel factor and c-kit is essential for the development of hematopoietic, gonadal and pigment stem cells. c-kit positive cells are a subset of CD34+ hematopoietic precursor cells and it is expressed on 5-10% of total adult bone marrow cells.
• Formulation: PBS containing 0.02% sodium azide (NaN3) as preservative and EIA grade BSA as a stabilizing protein to bring total protein concentration to 4-5 mg/ml.
  Label: Biotin
  State: Liquid purified Ig fraction
• Concentration: 0,1 mg/ml
• Purification: Affinity chromatography on Protein G
• Conjugation: Biotin
• Gene Name: Mus musculus KIT proto-oncogene receptor tyrosine kinase (Kit), transcript variant 2
• Database Link:
  Entrez Gene 16590 Mouse
• Synonyms: SCFR, KIT
• Note: Protocol: FLOW CYTOMETRY ANALYSIS:
  Method:
  1. Prepare a cell suspension in media A. For cell preparations, deplete the red blood cell population with Lympholyte®-M (cell separation medium).
  2. Wash 2 times.
  3. Resuspend the cells to a concentration of 2x10e7 cells/ml in media A. Add 50 μl of this suspension to each tube (each tube will then contain 1 x 10e6 cells, representing 1 test).
  4. To each tube, add 0.2-0.1 mg of this antibody per 10e6 cells.
  5. Vortex the tubes to ensure thorough mixing of antibody and cells.
  6. Incubate the tubes for 30 minutes at 4°C.
  7. Wash 2 times at 4°C.
  8. Add 100 μl of secondary antibody (Streptavidin-PE) at a 1/250 dilution.
  9. Incubate tubes at 4°C for 30 - 60 minutes (It is recommended that tubes are protected from light since most fluorochromes are light sensitive).
  10. Wash 2 times at 4°C.
  11. Resuspend the cell pellet in 50 μl ice cold media B.
  12. Transfer to suitable tubes for flow cytometric analysis containing 15 μl of propidium iodide at 0.5 mg/ml in PBS. This stains dead cells by intercalating in DNA.
  Media:
  A. Phosphate buffered saline (pH 7.2) + 5% normal serum of host species + sodium azide (100 μl of 2M sodium azide in 100 mls).
  B. Phosphate buffered saline (pH 7.2) + 0.5% Bovine serum albumin + sodium azide (100 μl of 2M sodium azide in 100 mls).
  Results:
  Tissue Distribution by Flow Cytometry Analysis:
  Mouse Strain: C3H/He
  Cell Concentration: 1x10e6 cells per test
  Antibody Concentration Used: 0.2 μg/10e6 cells
  Isotypic Control: Biotin Rat IgG2a
  Strain Distribution by Flow Cytometry Analysis:
  Cell Concentration: 1x10e6 cells per test
  Antibody Concentration Used: 0.2 mg /10e6 cells
  Strains Tested: AKR, BALB/c, C3H/He,
  Positive: AKR, BALB/c, C3H/He,
  Negative: none