Molecular Techniques and Methods

Unlabeled Protein Purification by
Immunoprecipitation with Antibody-Sepharose

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

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

Immunoprecipitation of unlabeled antigen followed by visualization with silver staining eliminates radiolabeling. Protein antigens present in greater than 104 copies per eukaryotic cell may be detected by immunoprecipitation of unlabeled cell lysates with antibody-Sepharose followed by SDS-PAGE and silver staining. If antigen is eluted from beads in SDS lacking reducing agents, little antibody is coeluted. To control for antibody eluting from the beads, a control of MAb-Sepharose incubated with mock lysate should be run simultaneously. A control of lysate incubated with nonspecific MAb-Sepharose should also be run.




MATERIALS AND SOLUTIONS

Lysis Buffer (1 liter)
0.5 % Triton X-100 ------------------------------ 10 ml of 50% Triton X-100
5 mM Iodoactamide ------------------------------ 925 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.




PROCEDURES

1. Incubate 5 x 107 cells/ ml in Lysis Buffer for 1 hour at 4oC.

2. Centrifuge 15 min at 3,000g and 4oC to remove nuclei.
Save the supernatant.

3. Centrifuge 1 hour at 200,000g. Save the supernatant.
Supernatants may also be prepared by microcentrifuging 30 min at 10,000g.

4. Preclear the lysate with 50 ul of activated, quenched Sepharose or nonspecific Ig-Sepharose/ ml antigen by gently shaking 1 hour at 4oC.

5. Centrifuge 5 min at 200g and save the supernatant. Preclearing removes nonspecifically absorbing material.

6. Mix 25 ul of a 1:1 (v/v) slurry of monoclonal antibody-Sepharose suspended in Dilution Buffer per ml of lysate and gently shake for 1 hour at 4oC.
  • The antibody coupled to Sepharose is antigen-specific.
  • 2 mg antibody/ ml Sepharose is coupled, and the amount can be estimated.

    7. Wash the Ab-Sepharose with 1 ml of the buffers listed below.
  • After each wash, microcentrifuge 5 seconds at high speed.
  • 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
    0.1% Triton X-100 in TSA Solution
    Second Wash
    0.1% Triton X-100 in TSA Solution
    Third Wash
    TSA Solution
    Fourth Wash
    0.05 M Tris-HCl (pH 6.8)


    8. Elution of antigen from Sepharose should be done under nonreducing conditions, since antibody is eluted from the beads under reducing conditions giving background staining. Theoretically only 1 of the 4 chains in an IgG antibody molecule covalently linked to the Sepharose, but interchain disulfide bonds keep all the chains linked under nonreducing conditions.

    9. For SDS-PAGE analysis of immunoprecipitates:
  • Add 20 to 50 ul of SDS/ sample buffer without 2-mercaptoethanol.
  • Do not vortex, since Sepharose sticks to sides of tubes above buffer level.
  • Cap tubes securely.
  • Heat 5 min at 100oC.
  • Microfuge for 5 second to pellet the Sepharose.
  • Remove the supernatant and apply to SDS-PAGE directly (non-reducing) or after incubaton for 1 hour at 37oC with 5% 2-mercaptoethanol (reducing).
  • Load the mixture into a gel lane and analyze by SDS-PAGE.
  • Detect antigen by Silver Staining.




    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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