Molecular Techniques and Methods

Radiolabeled-Protein Purification by
Immunoprecipitation with Antibody-Sepharose

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Immunoprecipitation can be used to precipitate selectively a given protein antigen from a complex protein mixture provided that specific antibodies directed against the protein are available.

Immunoprecipitation consists of multiple ordered steps.
(a) Lysing with detergent, if the protein to be immunoprecipitated is membrane-bound.
(b) Binding of a given protein antigen to an antibody.
(c) Precipitating the antibody-protein complex.
(d) Washing the precipitate.
(e) Dissociating the protein from the immune complex.
(f) The dissociated protein can be analyzed by electrophoretic methods.

This protocol relies on the formation of an insoluble immune complex between a protein antigen and an antigen-specific monoclonal (or polyclonal) antibody bound to Sepharose.




MATERIALS AND SOLUTIONS

Surface (or Biosynthetically)-Radiolabeled Cells


Lysis Buffer (1 liter)
1 % Triton X-100 --------------------------------- 20 ml of 50% Triton X-100
1 % Bovine hemoglobin --------------------------- 10 g
1 mM Iodoactamide ------------------------------ 185 mg
Aprotinin (Trypsin inhibitor) ----------------------- 200 U
1 mM PMSF ------------------------------------- 100 ml of 10 mM PMSF
Add TSA Solution to make a final volume of ---- 1 L
Store at 4oC.


Tris/ Saline/ Azide (TSA) Solution (1 liter)
0.01 M 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 H2O to make a final volume of ----- 1 L
Store at 4oC.


Cyanogen-Activated, Quenched Sepharose


Dilution Buffer (1 liter)
0.1% Triton X-100 ------------------------------- 2 ml of 50% Triton X-100
0.1% Bovine hemoglobin ------------------------- 1 g
Add TSA Solution 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




PROCEDURES

1. Incubate cells, surface-labeled with 125I or biosynthetically labeled with [35S]methionine or 3H- and 14C-labeled amino acids (5 X 107 Cells/ ml), in Lysis Buffer for 1 hr at 4oC.

2. Centrifuge the lysate 10 min at 3,000g and 4oC to remove nuclei.
Collect the supernatant.

3. Centrifuge the supernatant 1 hour at 200,000g and 4oC. Save the supernatant.
  • Supernatants may also be prepared by microcentrifugation for 30 min.

    4. The supernatant must be used within several days or stored at -80oC.
  • 125I-labeled antigen is stable 2 months at - 80oC, and the other isotopes (14C, 3H, and 35S) are stable 2 year.

    5. Preclear the lysate to be used in one batch by adding 10 ul of cyanogen-activated, quenched Sepharose or nonspecific antibody-Sepharose per 200 ul lysate.

    6. Shake 2 hour with an orbital shaker at room temperature or overnight at 4oC.

    7. Centrifuge 1 min at 200g and 4oC, and save the supernatant.
  • Preclearing removes nonspecifically absorbing material. A nonspecific antibody is an antibody directed against an unrelated protein, and it must not cross-react with the protein which is being immunoprecipitated.

    8. Precoat 1.5-ml microcentrifuge tubes by filling with Lysis Buffer 10 min at room temperature. Remove the solution by aspiration. Precoating prevents antigen absorption to the tube.

    9. Add 105 -106 cpm of radiolabeled (125I or 35S-) antigen to a precoated tube and bring the volume to 200 ul with Dilution Buffer.

    10. The recommended amount of radioactivity is appropriate for eukaryotic cells with > 1,000 molecules of antigen/ cell, and it is assumed that detection on slab gels of 125I-labeled proteins will be carried out with enhancing screens and 35S with fluorography.

    11. Add 10 ul of a 1:1 (vol/ vol) slurry of antibody-Sepharose in Dilution Buffer and shake 1.5 hour on an orbital shaker at 4oC.

    12. The antibody coupled to Sepharose is antigen-specific. 2 mg antibody/ ml Sepharose is coupled. Shaking must be vigorous enough to suspend the Sepharose. Shaking may be extended to 3 hour; longer periods may increase background.

    13. Wash the antibody-Sepharose with 1 ml of the buffers listed below.
  • After each wash, centrifuge 1 min at 200g or microcentrifuge 5 seconds.
  • Carefully aspirate the supernatant with a fine-tipped Pateur pipet and leave 10 ul of fluid above the pellet.
  • After the fourth wash, centrifuge again to bring down any residual drops on side of tube, aspirate, and leave 10 ul over pellet.

    First Wash
    1 ml of Dilution Buffer
    Second Wash
    1 ml of Dilution Buffer
    Third Wash
    1 ml of TSA Solution
    Fourth Wash
    1 ml of 0.05 M Tris-HCl (pH 6.8)


    14. Protein antigen can be eluted with 1 ml of Triethanolamine-NaCl Solution.

    15. For SDS-PAGE analysis of the immunoprecipitate: Add 20 to 50 ul of SDS/ sample buffer.
  • Do not vortex, since Sepharose may stick to side of tube above buffer level.
  • Incubate 5 min at 100oC.
  • Load the mixture into a gel lane and analyze by SDS-PAGE.
  • Detection of 125I-labeled proteins is carried out by autoradiography.




    NOTES

  • Reaction between antibody and antigen can be extended overnight, but this may increase the background when using antibody-bound Sepharose. Antigen-antibody complexes should not be left overnight in the midst of washing, since significant dissociation may occur.





  • KIT INFORMATION




    REFERENCES



  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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