Overview

  • Product name

    Protein G Sepharose® Column
  • Sample type

    Cell culture supernatant, Serum, Cell culture media, Ascites Fluid
  • Product overview

    Contents:


    Ready-to-use pre-packed columns of 1 mL or 5 mL bead volume in PBS with 0.02% sodium azide.


     


    Features:
    High binding capacity: Binding of IgG >20 mg human or rabbit IgG/mL Protein G Sepharose®.
    Minimal leaching of the ligand
    Flow Rate Tested*: 2.07 mL/min.
    *Test condition: Calculations based on the time required to pass 18 mL of water through 2 mL settled beads (column diameter 1.5 cm).
    Usage: Reusable for up to 10 times without significant loss of binding capacity.


    Store column at 4°C.


    The beads may be damaged above 40°C.


    DO NOT FREEZE.


    Wash beads 3 times with 3x bead volume of desired buffer before use.


     


    Applications:
    - 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 and the Fab region is available for binding antigen.


    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.


    Buffers:


    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


    Sepharose is a registered trademark of GE Healthcare

  • Notes

    Protein G Sepharose® Columns are prepared by covalently coupling recombinant Protein G to 6% cross-linked Sepharose® beads. Protein G is a genetically engineered protein containing three IgG-binding regions of native Protein G. The cell wall binding region, albumin binding region and other non-specific regions have been eliminated from the recombinant Protein G to ensure maximum specific IgG binding. The coupling technique is optimized to give a higher binding capacity for IgG and minimum leaching of recombinant Protein G. The IgG binding capacity of Protein G Sepharose® Column is >20 mg of human or rabbit IgG per mL of wet beads. Protein G Sepharose® Column display high chemical and physical stability as well as high flow rate, hydrophilicity and high gel strength. This product can be used for IgG purification and immunoprecipitation.

  • Tested applications

    Suitable for: IP, Purificationmore details

Properties

Applications

Our Abpromise guarantee covers the use of ab193260 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IP Use at an assay dependent concentration.

Isolation of antibody/antigen complexes in immunoprecipitation experiments, since only the Fc region is involved in antibody binding and the Fab region is available for binding antigen.

Purification Use at an assay dependent concentration.

Purification of monoclonal and polyclonal antibodies from culture media, serum, ascites fluid or hybridoma supernatants.

References

ab193260 has not yet been referenced specifically in any publications.

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