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The principle and method of immunoprecipitation (IP)

Immunoprecipitation (IP) is a method to isolate a specific antigen from a mixture, using the antigen-antibody interaction. Antigens isolated by IP are analyzed by SDS-PAGE or Western blotting.

The principle

Principle of IP
In IP, an antibody is added first to a mixture containing an antigen, and incubated to allow antigen-antibody complexes to form.
Subsequently, the antigen-antibody complexes are incubated with an immobilized antibody against the primary antibody (secondary antibody) or with protein A/G-coated beads to allow them to absorb the complexes.
The beads are then thoroughly washed, and the antigen is eluted from the beads by an acidic solution or SDS.

If suitable antibody is not available, the target molecule is fused to a His tag or other tags by recombinant DNA techniques, and immunoprecipitated using an antibody to the tag (pull-down assay).
[Related topics] Pull-down assay experiments using Tagged protein purification kits

AZ's point
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.


Tips for efficient IP

The diagram on the right illustrates the structures of antigen-antibody complexes formed with a monoclonal versus polyclonal antibody as the primary antibody.

Antigen-antibody complexWhen 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.

When using a polyclonal antibody, an excess of primary antibody, relative to the antigen, will prevent the formation of oligomeric complexes. Therefore, these concentrations should be optimized by testing different ratios.


Comparison of primary antibodies for IP
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.


Beads for immobilization

Agarose beads and magnetic beads are commonly used.
Agarose beads have a porous, mesh-like structure, and antibodies can diffuse and bind to the internal matrix of the beads, which provides high binding capacity.
Magnetic beads are simple spheres, providing ease of handling and short processing time. With appropriate coating, background can be reduced. However, binding capacity may not be high enough for some applications, and the cost is higher than the alternative using agarose beads.
Magnetic agarose beads provide ease of handling (when used with a magnet) and a high binding capacity.

Comparison of beads used for IP
Agarose beads Magnetic beads Magnetic agarose beads
Diameter (MBL products)
Approx. 100 µm Approx. 1.6 µm Approx. 50 µm
Diagram of the bead structure and antibody coat
Actual images
IgG binding capacity per matrix High Moderate High
Likelihood of sample loss Sample loss may occur during the washing step. Sample loss is negligible because the beads are pelleted using a magnet. Sample loss is minimal because the beads are pelleted using a magnet.
Centrifugation Necessary Unnecessary Unnecessary
Magnetic rack Unnecessary Necessary Necessary
Visibility of the beads Poor Excellent Excellent
Other features Inexpensive Easy to disperse; useful for screening, etc. Less expensive and provides a higher yield than magnetic beads.

[Related topics] Smart-IP tag-antibodies conjugated with magnetic beads or magnetic agarose

Detergents

Appropriate concentrations of salt and non-ionic detergent are commonly used in IP to reduce non-specific binding (protein-protein and protein-bead interactions). Pilot experiments should be performed with detergents, which may reduce the affinity of the antibody, especially when using a monoclonal antibody.

Protease inhibitors

The target protein and antibody are subject to degradation by protein-degrading enzymes (proteases) in samples such as cell lysates and tissue extracts. To prevent proteolytic degradation, protease inhibitors are included. When the type of proteases in samples are known or predicted, specific inhibitors are used. When proteases are unknown, a combination of multiple small molecule inhibitors, such as PMSF and EDTA, are used.

Elution condition

In most applications, proteins are eluted in SDS sample buffer containing a reducing agent, such as 2-mercaptoethanol(2-ME). In addition to the target protein, the antibodies used for IP are co-eluted, which should be considered when performing Western blotting or mass analysis.


Procedure

IP (with agarose beads) ※An example performed at MBL
Step-by-step procedure
Incubation with a primary antibody
Antigen-antibody complexes
Add 500 µL of protein extract and 2-10 µg of the primary antibody to a 1.5-mL tube.
IP - incubation with a primary antibody
Incubate at 4°C for 1 hour-overnight with shaking on a rotator. Rotator
Incubation with a secondary antibody
Secondary antibody-coated agarose beads
Add secondary antibody-coated (or protein A/G-coated) agarose beads.
IP - incubation with a secondary antibody
Incubate at 4°C for 1 hour-overnight on a rotating shaker.
Washing
IP washing
Pellet the agarose beads by centrifugation, and remove the supernatant by aspiration without disturbing the beads.
Remove the supernatant by aspiration
Add 1 mL of ice-cold lysis buffer or washing buffer, mix, and centrifuge. Remove the supernatant by aspiration without disturbing the beads. Repeat 3-4 times.
Add of ice-cold lysis buffer
<When performing SDS-PAGE or Western blotting>
Add 50 µL of 2x SDS sample buffer containing 2-mercaptoethanol (2-ME) and heat for 5 minutes to elute the target protein from the beads. Centrifuge, and use the supernatant for SDS-PAGE.
IP - elute with SDS sample buffer
※The antibodies are co-eluted, which should be taken into account when analyzing the data.
Add of 2x SDS sample buffer
<When extracting the target protein while preserving its activity and native conformation (pull-down assay)>
Elute the target protein in neutral pH using an elution peptide and avoid harsh conditions, such as acidic and alkaline solutions.
[Related topics] Pull-down assay experiments using Tagged protein purification kits


>>The principle and method of Western blotting (WB)
>>The principle and method of polyacrylamide gel electrophoresis (SDS-PAGE)

[Related topics] Links to IP-related products
Primary antibodies suitable for IP (excluding anti-tag antibodies) List of monoclonal primary antibodies
List of polyclonal primary antibodies
List of antibody-coated agarose beads
List of antibody-coated magnetic beads
List of antibody-coated magnetic agarose beads
Anti-tag antibodies suitable for IP List of monoclonal anti-tag antibodies
List of polyclonal anti-tag antibodies
List of anti-tag antibody-coated agarose beads
List of anti-tag antibody-coated magnetic beads
List of anti-tag antibody-coated magnetic agarose beads
Pull-down assay-related kits,
gels and elution peptides
List of tagged protein purification kits
List of tagged protein purification gel and elution peptide sets
Others Protein G-coated magnetic beads
Magnetic racks
For analysis of immunoprecipitated proteins HRP-DirecT series

Co-eluted antibody is not detected with HRP-labeled antibodies in Western blotting of immunoprecipitated proteins. It is especially useful when the target protein band overlaps with the heavy or light chain band. Analysis time is also reduced.








Related links

Fractionation and purification of proteins