Ly6g Rat Monoclonal Antibody [Clone ID: RB6-8C5]

CAT#: CL046P

Ly6g rat monoclonal antibody, clone RB6-8C5, Purified

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Specifications

Product Data

• Clone Name: RB6-8C5
• Applications: CT, FC, IHC, WB
• Recommended Dilution: This antibody is suitable for studies of myeloid differentiation stages and their regulations by cytokines. Applications include Flow Cytometry (1,2,3) complement-mediated depletion (4), Western blot staining (5) and both Frozen and Paraffin Sections (6).
• Reactivities: Mouse
• Host: Rat
• Isotype: IgG2b
• Clonality: Monoclonal
• Specificity: This monoclonal antibody reacts with the myeloid differentiation antigen Gr-1. (1,2).
  This 25-30 kDa cell surface antigen is expressed on myeloid cells but not lymphoid or erythroid cells. The expression of the Gr-1 antigen increases with granulocyte maturation (3) as shown by the distinct populations of bone-marrow cells this monoclonal antibody labels: negative, low positive and high positive. Expression is transient on cells of monocytic lineage (3).
• Formulation: PBS
  State: Purified
  State: Liquid purified IgG fraction
  Preservative: 0.02% Sodium Azide
• Concentration: lot specific
• Purification: Protein G Chromatography
• Conjugation: Unconjugated
• Storage: Store undiluted at 2-8°C for one month or (in aliquots) at -20°C for longer.
  Avoid repeated freezing and thawing.
• Stability: Shelf life: one year from despatch.
• Gene Name: lymphocyte antigen 6 complex, locus G
• Database Link:
  Entrez Gene 546644 Mouse
  UniProt ID P35461
• Background: The Gr-1 antigen is primarily a marker of myeloid differentiation. In the bone marrow the level of Gr-1 expression is low on immature myeloblasts and increases as the myeloid cells mature to granulocytes. Gr-1 is also expressed on macrophages and transiently on differentiating monocytes. Expression of Gr-1 on a subpopulation of lymphocytes has also been reported.
• Synonyms: Gr-1 Granulocyte marker
• Note: Protocol: FLOW CYTOMETRY ANALYSIS:
  
  Method:
  1. Prepare a cell suspension in media A. For cell preparations, deplete the red blood cell population with 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 0.1-0.2 µg* of CL046P.
  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 FITC Goat anti-rat IgG (H+L) .
  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:
  Mouse Strain: BALB/c
  Cell Concentration: 1x10e6 cells per test
  Antibody Concentration Used: 0.1 µg/10e6 cells
  Isotypic Control: Rat IgG2b
  
  Cell Source-Percentage of cells stained above control:
  Thymus: 2.0%
  Whole Blood Monocytes: 79.1%
  Bone Marrow Macrophages: 87.5%
  
  N.B. Appropriate control samples should always be included in any labelling studies.
  * For optimal results in various applications, it is recommended that each investigator determine dilutions appropriate for individual use.
  
  Strain Distribution by Flow Cytometry Analysis:
  Cell Concentration: 1x10e6 cells per test
  Antibody Concentration Used: 0.1 µg/10e6 cells
  Strains Tested: BALB/c, C57BL/6, CBA, C3H/He, AKR
  Positive: BALB/c, C57BL/6, CBA, C3H/He, AKR
  Negative: none