CXCR4 (extracell. Dom.) (N-term) Rat Monoclonal Antibody [Clone ID: A145]

CAT#: AM26473AF-N

CXCR4 (extracell. Dom.) (N-term) rat monoclonal antibody, clone A145, Azide Free

Specifications

Product Data

• Clone Name: A145
• Applications: FC
• Recommended Dilution: Flow cytometry: 10-20 μg/ml (final concentration). For details see protocols below.
• Reactivity: Human
• Host: Rat
• Isotype: IgG1
• Clonality: Monoclonal
• Immunogen: Cos-1 cells transfected with CXCR4 gene
• Specificity:

  The antibody recognizes the N-terminal, extracellular domain of the CXCR4 protein.

• Formulation: PBS containing 50% glycerol. Contains no preservatives.
  State: Azide Free
  State: Liquid Ig fraction
• Concentration: lot specific
• Purification: Ammonium sulfate precipitation followed by gel filtration through Superdex 200 in PBS
• 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.

• Database Link:
  Entrez Gene 7852 Human
• Synonyms: CXC-R4, CXCR-4, Fusin, LCR1, FB22, NPYRL, HM89, SDF1 receptor, LESTR
• Note: This product was originally produced by MBL International.
  
  Protocol:

  Flow cytometric analysis for floating cells
  
  Protocol 1
  
  1) Wash the cells 3 times with washing buffer [PBS containing 2% fetal calf serum (FCS) and 0.1% NaN3].
  2) Resuspend the cells with washing buffer (5x10e6 cells/mL).
  3) Add 50 µL of the cell suspension into each tube, and centrifuge at 500xg for 1 minute at room temperature (20~25oC). Remove supernatant by careful aspiration.
  4) Add 10 µL of normal goat serum containing 1 mg/mL normal human IgG and 0.1% NaN3 to the cell pellet after tapping. Mix well, and incubate for 5 minutes at room temperature (20~25 oC).
  5) Add 30 µL of the anti-CXCR4 monoclonal antibody (10-20 µg/mL) diluted with the washing buffer. Mix well, and incubate for 30 minutes at room temperature (20~25 oC).
  6) Add 1 mL of the washing buffer followed by centrifugation at 500xg for 1 minute at room temperature (20~25oC). Remove supernatant by careful aspiration.
  7) Add 30 µL of secondary antibody (1:40 FITC conjugated anti-Rat IgG) diluted with the washing buffer. Mix well and incubate for 15 minutes at room temperature (20~25oC).
  8) Add 1 mL of the washing buffer followed by centrifugation at 500xg for 1 minute at room temperature (20~25oC). Remove supernatant by careful aspiration.
  9) Resuspend the cells with 500 µL of the washing buffer and analyze by a flow cytometer.
  
  Protocol 2

  1) Wash the cells 3 times with washing buffer [PBS containing 2% fetal calf serum (FCS) and 0.1% NaN3].
  2) Resuspend the cells with PBS containing 25% normal goat serum, 1 mg/mL normal human IgG and 0.1% NaN3 (5x106 cells/mL).
  3) Add 20 µL of the anti-CXCR4 monoclonal antibody (50 µg/mL) diluted with the washing buffer into each tube.
  4) Add 50 µL of the cell suspension into each tube. Mix well, and incubate for 30 minutes at room temperature (20~25 oC).
  5) Add 1 mL of the washing buffer followed by centrifugation at 500xg for 1 minute at room temperature (20~25oC). Remove supernatant by careful aspiration.
  6) Resuspend the cells with 50 µL of the washing buffer.
  7) Add 20 µL of secondary antibody (1:10 FITC conjugated anti-Rat IgG) diluted with the washing buffer into each tube. Mix well and incubate for 15 minutes at room temperature (20~25oC).
  8) Add 1 mL of the washing buffer followed by centrifugation at 500xg for 1 minute at room temperature (20~25oC). Remove supernatant by careful aspiration.
  9) Resuspend the cells with 500 µL of the washing buffer and analyze by a flow cytometer.