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Protein A Sepharose Column Bead

IP, Purif Protein A Sepharose beads 90 µm
Catalog No. ABIN412448
$145.00
Plus shipping costs $45.00
1 mL ABIN412448
1 mL ABIN412448
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  • Target
    Reactivity
    Staphylococcus aureus
    Application
    Immunoprecipitation (IP), Purification (Purif)
    Purpose
    Protein A Sepharose Columns can be used for IgG purification and immunoprecipitation
    Characteristics
    Protein A Sepharose is prepared by covalently coupling recombinant Protein A to 6 % cross-linked sepharose beads. The coupling technique is optimized to give a high binding capacity for IgG. The capacity of IgG binding could be up to 25 mg of human IgG per mL of wet bead.
    BINDING CAPACITY: Binding of IgG ≥ 16 mg human or rabbit IgG/ml Protein A-Sepharose.
    FLOW RATE TESTED: 2.07 ml/min
    USAGE: Reusable for up to 10 times without significant loss of binding capacity.
    Bead Ligand
    Protein A
    Bead Matrix
    Sepharose beads
    Bead Size
    90 µm
  • Target
  • Application Notes
    - Purification of monoclonal and polyclonal antibodies from culture media, serum, ascites fluid or hybridoma supernatants.
    - Isolation of antibody/antigen complexes in immunoprecipitation experiments, since only the Fc region is involved in antibody binding andthe Fab region is available for binding antigen.
    Comment

    Protein A-Sepharose beads are prepared by covalently coupling recombinant Protein A to 6% cross-linked Sepharose beads. The coupling technique is optimized to give a higher binding capacity for IgG & minimum leaching of recombinant Protein A. The IgG binding capacity of Protein A-Sepharose is ≥ 16 mg human or rabbit IgG per ml of wet beads.
    BINDING CAPACITY: Binding of IgG ≥ 16 mg human or rabbit IgG/ml Protein A-Sepharose.
    FLOW RATE TESTED: 2.07 ml/min
    USAGE: Reusable for up to 10 times without significant loss of binding capacity.

    Assay Procedure

    PROTOCOL EXAMPLE (ANTIBODY PURIFICATION):
    1. Carefully pack the column avoiding air bubbles.
    2. Equilibrate the column with 5X resin bed volume of Binding Buffer & allow the buffer to drain through the column. Do not let the resin bed dry.
    3. Dilute serum sample with Binding Buffer (1:1 ratio).
    4. Mix well the diluted serum sample. Make sure there are no bubbles in the sample solution.
    5. Apply the diluted sample onto the column. Do not let the resin bed dry.
    6. Collect the flow-through.
    7. Reapply the flow-through to the column & collect the sample. Repeat 4 times.
    8. Wash the column 4 – 5 times with 5X volume of Binding Buffer containing 0.5 M NaCl.
    9. Wash the column 4 - 5 times with Binding Buffer.
    10. Elute antibodies with Elution Buffer ~3-5X resin bed volume.
    11. Collect fractions using micro centrifuge tube. Immediately neutralize the eluted fractions by adding 100 µl of 1 M Tris, pH 9.0 per ml of eluate.
    12. Assay protein concentration by measuring the absorbance at 280 nm and combine the fractions with highest absorbance. 1 OD280 = 0.73 mg/ml IgG.
    13. To regenerate/store column:
    a. Wash with 5 volumes of Elution Buffer.
    b. Wash with 5 volumes of distilled water.
    c. Store column in 20 % Ethanol/H2O at 4°C

    Restrictions
    For Research Use only
  • Format
    Liquid
    Buffer
    Ready-to-use pre-packed columns of 1 ml or 5 ml bead volume in PBS with 0.02% sodium azide, >5mg Protein A/ml Sepharose beads.
    Binding Buffer: PBS/TBS/0.15 M sodium chloride in 50 mM sodium borate, pH 8.0
    Elution Buffer: 0.1 M citric acid, pH 2.75
    Storage
    4 °C
    Expiry Date
    12 months
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