24 Protein Techniques
371
lmmunoprecipitation
Immunoprecipitation involves the use of antihoclies in solution to bind and
precipitate specific antigens. The advantages of this technique compared to
Western blotting include: relatively mild denaturing conditions are used,
more native conformations are detected, the use of radiolabeled material is
permitted, high specificity and efficiency, and further biochemical analysis
of isolated proteins is possible. lts disadvantages include: dependence on
the ability to radiolabel the antigen of interest, relatively high avidity antihodies are required, and the method is relatively time consuming.
Immunoprecipitation involves four basic processes: binding of the antigen
to the antibody; precipitation of the antigen-antibody complex, washing;
and elution.
Binding of the antigen to the antibody: Binding occurs equally well at any
temperature but is generally carried out at 4°C to avoid proteolytic degradation. The precipitation should be performed on the same day as the labeling, since freeze-thawing of the antigen-containing mixture may cause
protein denaturation and aggregation. If the antigen must be frozen, it
should be snap frozen in an ethanol-dry ice bath and stored at -70°C. Antibody should be present in large excess; this usually requires 5-50 111 of polyclonal sera or 20-200 111 ofhybridoma supernatant. The duration of incubation with the primary antibody is often not critical (30-60 min). Incubation
is carried out at physiological salt concentration and pH. Occasionally, individual monoclonal antihoclies may precipitate in the cold or at low ionic
strength.
Precipitation of the antigen antibody complex: Although there are two
methods generally available for the precipitation of antigen-antibody complexes, equivalence complexes and solid-phase immunoadsorption, only
the latter is currently in widespread use. Solid-phase immunoadsorption
involves the selective absorption of the antigen-antibody complex to an affinity matrix. Complexes are often adsorbed onto protein A-Sepharose (9).
ProteinAis a bacterial proteinthat binds the Fe domain of many IgGs, and
some IgMs and IgAs. lt has high affinity to most but not all IgG isotypes.
Protein A binds immunoglobulins from certain species better than others:
human, pig > mouse > rabbit, goat, sheep » rat. A similar protein, protein G,
has a broader species specificity and binds certain rat IgGs well. Solid-phase
methods have the advantage that there is less IgG in the final pellet which
results in less overloading of the SDS-PAGE gel and less nonspecific pre-
371
lmmunoprecipitation
Immunoprecipitation involves the use of antihoclies in solution to bind and
precipitate specific antigens. The advantages of this technique compared to
Western blotting include: relatively mild denaturing conditions are used,
more native conformations are detected, the use of radiolabeled material is
permitted, high specificity and efficiency, and further biochemical analysis
of isolated proteins is possible. lts disadvantages include: dependence on
the ability to radiolabel the antigen of interest, relatively high avidity antihodies are required, and the method is relatively time consuming.
Immunoprecipitation involves four basic processes: binding of the antigen
to the antibody; precipitation of the antigen-antibody complex, washing;
and elution.
Binding of the antigen to the antibody: Binding occurs equally well at any
temperature but is generally carried out at 4°C to avoid proteolytic degradation. The precipitation should be performed on the same day as the labeling, since freeze-thawing of the antigen-containing mixture may cause
protein denaturation and aggregation. If the antigen must be frozen, it
should be snap frozen in an ethanol-dry ice bath and stored at -70°C. Antibody should be present in large excess; this usually requires 5-50 111 of polyclonal sera or 20-200 111 ofhybridoma supernatant. The duration of incubation with the primary antibody is often not critical (30-60 min). Incubation
is carried out at physiological salt concentration and pH. Occasionally, individual monoclonal antihoclies may precipitate in the cold or at low ionic
strength.
Precipitation of the antigen antibody complex: Although there are two
methods generally available for the precipitation of antigen-antibody complexes, equivalence complexes and solid-phase immunoadsorption, only
the latter is currently in widespread use. Solid-phase immunoadsorption
involves the selective absorption of the antigen-antibody complex to an affinity matrix. Complexes are often adsorbed onto protein A-Sepharose (9).
ProteinAis a bacterial proteinthat binds the Fe domain of many IgGs, and
some IgMs and IgAs. lt has high affinity to most but not all IgG isotypes.
Protein A binds immunoglobulins from certain species better than others:
human, pig > mouse > rabbit, goat, sheep » rat. A similar protein, protein G,
has a broader species specificity and binds certain rat IgGs well. Solid-phase
methods have the advantage that there is less IgG in the final pellet which
results in less overloading of the SDS-PAGE gel and less nonspecific pre-