Immunoprecipitation using Affinity Agarose Resins
The combined procedures of immunoprecipitation and SDS-PAGE can be a powerful tool to assess the amount and size of an antibody-reactive antigen present in a complex protein mixture. The basic protocol uses a primary antibody followed by a secondary antibody-agarose conjugate to immunoprecipitate the antigen.
Reagents Required
TBS: Use 10XTBS, pH 7.5 (1.0M Tris HCl, 1.5M NaCl). Dilute appropriate volume to 1X with dH2O. Store at RT up to one month.
Lysis Buffer: TBS, 1.0% of an appropriate detergent (i.e. Triton X-100), 1mg/ml BSA and an appropriate proteinase inhibitor.
Dilution buffer: Same as lysis buffer without proteinase inhibitor.
Agarose conjugates for lysate pre-treatment. Pre-adsorb lysate to remove non-specific binding to primary and secondary antibodies. Use agarose normal IgG from the same species as the primary antibody and the host secondary antibody. Prepare washed slurry at 1:1 using dilution buffer.
Primary Antibody
Control for primary antibody: For polyclonal antiserum, use nonimmune serum from the same species. For monoclonal antibodies, use the same isotype and purity.
Agarose conjugates for Immunoprecipitation: Use agarose secondary antibody conjugate against the same species as the primary antibody. Prepare washed slurry at 1:1 using dilution buffer.
Tris Buffer: Prepare 0.05M Tris buffer, pH 6.8.
2XSDS-PAGE Sample Buffer.
2-Mercaptoethanol
Procedure
1.Prepare lysate by incubating 5 x 10e7 cells in lysis buffer for 30 to 60 min on ice.
2.Vortex lysate and centrifuge for 10 min at 250 x g to remove nuclei. Retain supernate.
3.Clarify supernate by centrifugation for 30 min at 100,000 x g or microcentrifuge for 30 min at 10,000 x g.
4.Pretreat lysate to remove nonspecific protein binding by adding agarose conjugates. Use 10ul of control agarose per 200ul lysate. Shake for 1 hour at 4°C. Centrifuge at 200 x g. Save supernatant.
5.Add 200ul of pretreated lysate containing antigen to each of two microfuge tubes. Bring volume to 1ml with dilution buffer.
6.Add primary antibody to one tube. For polyclonal antiserum or ascites fluid use 0.5-5.0ul. For tissue culture supernatant use 10-100ul. To the second tube add an equivalent volume of control for primary antibody. Incubate on ice for 1 hour.
7.For immunoprecipitation add 50ul of agarose conjugate per tube. Mix with gentle shaking for 1 hour at 4°C.
8.Centrifuge tube 1 min at 200 x g or microcentrifuge for 5 seconds. Carefully remove the supernate with a pipette. Gently resuspend pellet in 1ml dilution buffer. Repeat wash. Follow with a wash in TBS and then a final wash in 0.5M Tris, pH 6.8.
9.Centrifuge again as above. Add 20-50ul of sample buffer. Mix and heat for 5 min at 100°C. Microcentrifuge briefly and apply supernate directly to nonreducing SDS-PAGE. If reducing conditions are desired, transfer the supernate to a new tube and add 5% 2-mercaptoethanol. Mix and heat as above.
10.Electrophorese protein mixture. Stain gel or immunoblot to visualize. Bands present will include polypeptides of antigen and antibodies used.