2. Immunoprecipitation
Immunoprecipitation (IP) is the technique of precipitating a
protein antigen out of solution using an antibody that
specifically binds to that particular protein.
This process can be used to isolate and concentrate a
particular protein from a sample containing many
thousands of different proteins.
Immunoprecipitation requires that the antibody be coupled
to a solid substrate at some point in the procedure.
Antigens isolated by IP are analyzed by SDS-PAGE or
Western blotting.
3. Types of immunoprecipitation
There are different types of immunoprecipitation:
Individual protein immunoprecipitation (IP): Involves using an antibody that is specific for a known
protein to isolate that particular protein out of a solution containing many different proteins.
Protein complex immunoprecipitation (Co-IP): Immunoprecipitation of intact protein complexes is
known as co-immunoprecipitation (Co-IP). Co-IP works by selecting an antibody that targets a known
protein that is believed to be a member of a larger complex of proteins.
Chromatin immunoprecipitation (ChIP): Chromatin immunoprecipitation (ChIP) is a method used to
determine the location of DNA binding sites on the genome for a particular protein of interest. This
technique gives a picture of the protein–DNA interactions that occur inside the nucleus of living cells or
tissues.
RNP immunoprecipitation targets ribonucleoproteins (RNPs): Live cells are first lysed and then the
target protein and associated RNA are immunoprecipitated using an antibody targeting the protein of
interest
4. General methods for immunoprecipitation:
Direct capture method
Antibodies that are specific for a particular protein (or group of proteins) are immobilized on a solid-phase substrate such
as superparamagnetic microbeads or on microscopic agarose (non-magnetic) beads.
The beads with bound antibodies are then added to the protein mixture, and the proteins that are targeted by the antibodies
are captured onto the beads via the antibodies; in other words, they become immunoprecipitated.
indirect capture method
The antibodies have not been attached to a solid-phase support yet. The antibodies are free to float around the protein
mixture and bind their targets.
As time passes, the beads coated in protein A/G are added to the mixture of antibody and protein. At this point, the
antibodies, which are now bound to their targets, will stick to the beads.
5. Steps of immunoprecipitation:
1. Lyse cells and prepare sample for immunoprecipitation.
2. Pre-clear the sample by passing the sample over beads alone or bound to an irrelevant antibody to soak up any
proteins that non-specifically bind to the IP components.
3. Incubate solution with antibody against the protein of interest. Antibody can be attached to solid support before
this step (direct method) or after this step (indirect method). Continue the incubation to allow antibody-antigen
complexes to form.
4. Precipitate the complex of interest, removing it from bulk solution.
6. Steps of immunoprecipitation:
5. Wash precipitated complex several times. Spin each time between washes when using agarose beads or place
tube on magnet when using superparamagnetic beads and then remove the supernatant. After the final wash,
remove as much supernatant as possible.
6. Elute proteins from the solid support using low-pH or SDS sample loading buffer.
7. Analyze complexes or antigens of interest. This can be done in a variety of ways:
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by gel staining.
SDS-PAGE followed by: gel staining, cutting out individual stained protein bands, and sequencing the proteins
in the bands by MALDI-Mass Spectrometry
Transfer and Western Blot using another antibody for proteins that were interacting with the antigen, followed
by detection using a chemiluminescent or fluorescent secondary antibody.
7. Important tips for efficient IP:
The use of an antibody with high binding specificity and affinity for the antigen is
critical for successful IP.
Antibodies raised against synthetic peptides and recombinant proteins often work well
in Western blotting but may not bind the antigens in their native conformation in
solution.
When using commercial antibodies, select the ones that are suitable for IP according to
product information. Also recognize the properties of both the antibody and antigen in
the literature and product information.
When using a monoclonal antibody as the primary antibody, adjust the concentration
so that: [secondary antibody] > [primary antibody] > [antigen].
An excess of primary antibody, relative to the secondary antibody, may compete with
antigen-antibody complexes for the secondary antibody, resulting in a lower yield of
recovery.
8. Monoclonal antibody Polyclonal antibody
Background
Background is low with an
appropriate antibody because
the antibody recognizes a
single antigen.
Background may be high if
some of the antibody
molecules have a low
specificity and bind to
proteins other than the target
protein.
Binding affinity
High-affinity monoclonal
antibody (dissociation
constant Kd<10-8
M) should be
used because low affinity
antibody may not form an
antigen-antibody complex in
solution.
Even if the affinity of
individual antibody molecules
is low, oligomeric antigen-
antibody complexes are
formed easily due to the
multivalent binding.
Stability of antigen-antibody
complexes
If the binding affinity of an
antibody is low,
simultaneously using several
high-specificity monoclonal
antibodies will allow
multivalent binding, resulting
in stable antigen-antibody
complexes.
Stable oligomeric complexes
are formed because reaction
between a polyclonal
antibody and an antigen is
multivalent.
Comparison of primary antibodies for IP
10. Application of immunoprecipitation:
Isolate / Detect Proteins of interest
Enrichment of low abundant proteins
Study protein-protein interaction and protein complexes
Identify unknown proteins in a protein complex
Verify protein expression in a specific tissue.
