Molecular Techniques and Methods

Protein Purification by
Immunoaffinity Column Chromatography

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

These protocols are established to isolate membrane-bound or soluble proteins from cells or homogenized tissue. Antibodies are coupled to Sepharose. High-molecular-weight antigens pass freely into and out of the pores and bind to antibodies covalently bound to the matrix. In order to elute the bound protein antigen from the immunoaffinity matrix, the antibody-antigen interaction is destabilized by brief exposure to high-pH or low-pH buffer.




MATERIALS AND SOLUTIONS

Antigen-Specific Monoclonal (or Polyclonal) Antibody


Dialysis Buffer (1 liter)
0.1 M NaHCO3 ------------------------------------ 8.4 g
0.5 M NaCl ---------------------------------------- 100 ml of 5 M NaCl
Add distilled Water to make a final volume of ------- 1 L
  • Store at 4oC


    Sepharose CL-4B
    (or Sepharose CL-6B for high-molecular-weight antigens)


    0.2 M Na2CO3 (1 liter)
    Na2CO3 -------------------------------------------- 21.2 g
    Add distilled Water to make a final volume of ------- 1 L
  • Store at 4oC


    Sepharose Activation Solution (123 ml)
    CNBr --------------------------------------------- 50 g
    Acetonitrile ---------------------------------------- 100 ml
  • Store at -20oC in a desiccator over silica.


    Tris/ Saline/ Azide (TSA) Solution (1 liter)
    0.002 M Tris-HCl (pH 8.0) ------------------------ 2 ml of 1 M Tris-HCl
    0.14 M NaCl -------------------------------------- 28 ml of 5 M NaCl
    0.025% NaN3 ------------------------------------- 250 mg
    0.5% Triton X-100 -------------------------------- 10 ml of 50% Triton X-100
    0.5% Sodium Deoxycholate ------------------------ 5 g
    Add distilled Water to make a final volume of ------- 1 L
  • Store at 4oC


    Lysis Buffer (100 ml)
    2% Triton X-100 ---------------------------------- 4 ml of 50% Triton X-100
    5 mM Iodoacetamide ----------------------------- 93 mg
    Aprotinin (Trypsin Inhibitor) ----------------------- 20 U
    1 mM PMSF -------------------------------------- 10 ml of 10 mM PMSF
    Add TSA Solution to make a final volume of ------- 100 ml
  • Store at 4oC


    Wash Buffer (1 liter)
    10 mM Tris-HCl (pH 8.0) ------------------------- 10 ml of 1 M Tris-HCl
    0.14 M NaCl -------------------------------------- 28 ml of 5 M NaCl
    0.025% NaN3 ------------------------------------- 250 mg
    Add distilled Water to make a final volume of ------- 1 L
  • Store at 4oC


    Tris-NaCl Buffer (pH8.0) (1 liter)
    50 mM Tris-HCl (pH 8.0) -------------------------- 50 ml of 1 M Tris-HCl
    0.1% Triton X-100 --------------------------------- 2 ml of 50% Triton X-100
    0.5 M NaCl ---------------------------------------- 100 ml of 5 M NaCl
    Add distilled Water to make a final volume of ------- 1 L
  • Store at 4oC


    Tris-NaCl Buffer (pH9.0) (1 liter)
    50 mM Tris-HCl (pH 9.0) -------------------------- 50 ml of 1 M Tris-HCl
    0.1% Triton X-100 --------------------------------- 2 ml of 50% Triton X-100
    0.5 M NaCl ---------------------------------------- 100 ml of 5 M NaCl
    Add distilled Water to make a final volume of ------- 1 L
  • Store at 4oC


    Triethanolamine-NaCl Solution (1 liter)
    50 mM Triethanolamine (pH11.5) ------------------- 7.5 g
    0.1 % Triton X- 100 -------------------------------- 2 ml of 50% Triton X-100
    0.15 M NaCl --------------------------------------- 30 ml of 5 M NaCl
    Add distilled Water to make a final volume of ------- 1 L
  • Store at 4oC


    Column Storage Solutions (1 liter)
    1 mM EDTA --------------------------------------- 2 ml of 0.5 M EDTA
    Gentamycin ----------------------------------------- 20 g
    Add distilled Water to make a final volume of ------- 1 L
  • Store at 4oC




    PROCEDURES

  • All steps should be carried out in a 4oC cold room or on ice.

    Preparation of an Immunoaffinity (Antibody-Sepharose) Column by Cyanogen Activation

    1. Dialyze 1-30 mg/ ml antibody against Dialysis Buffer at 4oC with three buffer changes during 24 hour. The volume of dialysis solution should be 500 times the volume of antibody solution. Use dialysis tubing with molecular weight cutoff 12,000-14,000.
  • Dialysis is performed to remove all small molecules containing free amino or sulfhydryl groups.

    2. Centrifuge 15 min at 100,000g and 4oC to remove aggregates.

    3. Measure the A280 of an aliquot of the solution and determine the concentration of the antibody (i.e. 1 mg IgG /ml = 1.44 A280).

    4. Dilute antibody with Dialysis Buffer to obtain the concentration of the antibody-Sepharose (5 mg/ml).

    5. Prepare the Sepharose by allowing the Sepharose CL-4B slurry to settle in a water and decant the supernatant.

    6. Weigh out the desired quantity of Sepharose CL-4B, assuming the density = 1.0.
  • Wash the Sepharose with 10 volume of distilled water by using Whatman No.1 filter paper in a Buchner funnel and an Erlenmeyer filtration flask attached to a water aspirator.

    7. Transfer Sepharose to 50 ml beaker and add an equal volume of 0.2 M Na2CO3.

    8. Activate Sepharose by adding 3.2 ml Sepharose Activation Solution per 100 ml Sepharose at room temperature.
  • Add Sepharose Activation Solution dropwise over 1 min, while slowly stirring the slurry with a magnetic stirrer in a fume hood.

    9. After 5 min, rapidly filter the activated Sepharose by using Whatman No.1 filter paper in a Buchner funnel and an Erlenmeyer filtration flask attached to a water aspirator.
  • Aspirate to semidryness.

    10. Wash with 10 vol of ice-cold 1 mM HCl.

    11. Wash with 2 vol of ice-cold 0.1 mM HCl.
  • Washing is most efficient if the wash solution is added evenly over the surface of the cake at about the same rate as the solution is removed by filtration.
  • Activated Sepharose is very unstable at the alkaline pH necessary for activation. It is much more stable in dilute HCl.

    12. Couple the antibody to activated Sepharose.
    Immediately transfer a weighed amount of Sepharose (density = 1.0) to a beaker.
    Add an equal volume of a solution of antibody dissolved in ice-cold Dialysis Buffer.

    13. Stir gently with a magnetic stirrer for 1 hour at room temperature or overnight at 4oC.

    14. Add 0.05 M glycine to saturate the remaining reactive groups on the Sepharose.
    Allow the slurry to settle.

    15. Centrifuge and measure the A280 of an aliquot of the supernatant.
    Compare absorbance to that of the A280 of the antibody solution in step 1 to determine the percentage coupling.

    16. Prepare an activated, quenched Sepharose precolumn (5 ml packed bed volume) and immunoaffinity column (5 ml, 5 mg antibody/ ml packed Sepharose) linked in series.

  • The activated, quenched Sepharose precolumn is prepared in an identical manner to the antibody-Sepharose column, except that antibody is not added during the coupling step.



    Preparation of the Lysate

    17. Suspend 50 g of cells or homogenized tissue at 1-5 x 108 cells/ ml of ice-cold TSA in a plastic beaker.

    18. Add an equal volume of ice-cold Lysis Buffer and stir at 4oC for 1 hour.

    19. Centrifuge 10 min at 4,000g and 4oC. Decant supernatant
  • This spin removes nuclei.
  • For purification of cytoplasmic protein antigens, it is not necessary to add detergents to the solutions and buffer used in subsequent steps.
  • For purification of membrane-bound protein antigens, add 0.2 vol of 5% Sodium deoxycholate, to the post-nuclear supernatant and swirl to mix for 10 min. Centrifuge in quickseal tubes at 150,000g and 4oC for 2 hour. Carefully remove the supernatant.



    Column Wash

    20. Attach Sepharose precolumn to immunoaffinity column.

    21. Wash the immunoaffinity column and precolumn with 10 column volumes of Wash Buffer.

    22. Wash the immunoaffinity column and precolumn with 5 column volumes of Tris-NaCl Buffer (pH 8.0).

    23. Wash the immunoaffinity column and precolumn with 5 column volumes of Tris-NaCl Buffer (pH 9.0).

    24. Wash the immunoaffinity column and precolumn with 5 column volumes of Triethanolamine-NaCl Solution.

    25. Wash the immunoaffinity column and attached Sepharose precolumn with 5 column volumes of Wash Buffer.



    Purification of the Protein Antigen

    26. Apply the lysate (supernatant from step 19) to the columns at a flow rate of 5 column volumes/ hour.

    27. Use chromatography columns (2 cm height x 2.5 cm diameter) filled with Sepharose to maximize flow rates.
    The flow rate is adjusted with a hydrostatic head of up to 250 cm.
    Sample loading can routinely take up to 2 days with no deleterious effect, but longer periods would suggest the column is clogged or the lysate is too viscous.
    The latter is usually due to the presence of DNA.

    28. Close the stopcocks on both columns and disconnect the precolumn from the immunoaffinity column.
    Open the stopcock of the immunoaffinity column and allow fluid above the top of the column to drain out until just above bed level.

    29. Wash with 10 column volumes of Wash Buffer.

    30. Wash with 5 column volumes of Tris-NaCl Buffer (pH 8.0).

    31. Wash with 5 column volumes of Tris-NaCl Buffer (pH 9.0).
    Some non-specifically bound proteins may be eluted at this step.

    32. Elute with 5 column volumes of Triethanolamine-NaCl Solution.

    33. Collect fractions into tubes containing 0.2 vol of 1 M Tris-HCl (pH 6.7), in order to neutralize the fractions collected.

    34. Wash the column in TSA.

    35. A column may be reused many times and remain active for several years after storage at 4oC in TSA.
    It is important to prevent the drying out of a column during storage.
    The use of Column Storage Solutions inhibits the growth of microorganisms.

    36. Analyze fractions for antigen. Aliquots of eluate fractions (50 ul) should be analyzed by SDS-PAGE and silver staining.




    NOTES

  • Binding capacities of antibody-Sepharose columns (coupled at 10 mg monoclonal antibody/ml Sepharose) have been found to be 2-20% of the theoretical binding capacity.

  • Detergent choice will affect the efficiency of solubilization and will influence whether multimeric membrane protein ensembles and interactions with peripheral membrane proteins are dissociated or remain intact. A number of non-ionic detergents may be substituted for Triton X-100. Brij-96, Brij-97, and Lubrol-PX are alkylpoly-oxyethylenes, which give low absorbance at 280 nm. Sodium deoxycholate has a much smaller micelle size than non ionic detergents and may be removed by dialysis.

  • Sodium deoxycholate is used because it is a more strongly dissociating detergent than Triton X-100. However, since sodium deoxycholate releases DNA from nuclei, it must be added to the lysate after the nuclei are removed.




    KIT INFORMATION




    REFERENCES

  • Ey RL, Prowse SJ, Jenkin CR (1978) Isolation of pure IgG1, lgG2a, and IgG2b immunoglobulins from mouse serum using protein A-Sepharose. lmmunochemistry 15:429-436.

  • Johnson P, Williams AF, Woollett GR (1985) Purification of membrane glycoproteins with monoclonal antibody affinity columns. In "Hybridoma Technology in the Biosciences and Medicine" (Ed. T.A. Springer) pp163-175. Plenum, New York.

  • Wilchek M, Miron T, Kohn J (1984) Affinity chromatography. Meth. Enzymol. 104:3-55.

  • Williams AF, Barclay AN (1986) Glycoprotein antigens of the lymphocyte surface and their purification by antibody affinity chromatography. In "lmmunological Methods in Biomedical Sciences" (Eds. DM Weir, LA Herzenberg, CC Blackwell, LA Herzenberg) pp22.1-22.4. Blackwell, Oxford.


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