Immunoprecipitation (IP) Protocol and FAQs

Immunoprecipitation (IP) is an experimental technique widely used in biological and biochemical studies. It aims to enrich and separate target proteins or their complexes from mixtures by specific antibodies. IP technology is usually used to study the interaction between proteins, protein expression, and protein modification.

The Principle of Immunoprecipitation

The core idea of immunoprecipitation is to use the specific binding of antibodies to target antigens. In the experiment, the antibody is first mixed with a cell lysate containing the target protein or other samples, and the antibody will bind to the target protein to form a complex. Subsequently, the antibody-protein complex was separated from the solution by adding appropriate protein A / G agarose beads or magnetic beads. The separated complex can be washed to remove non-specific binding substances, and finally detected by Western Blot and other techniques.

The Steps of Immunoprecipitation

Sample Preparation

Sample preparation is the first step of immunoprecipitation experiments. According to the needs of the experiment, the sample can be cell lysate, tissue extract or culture medium. During the preparation process, appropriate lysis buffers are usually used to release proteins from cells or tissues. At this time, the sample contains the target protein and possible non-target proteins, nucleic acids and other components.

Antibody Binding

Specific antibodies were added to the sample. Antibodies usually target specific epitopes of the target protein, so it can efficiently bind to the target protein. The antibody binds to the target protein to form an antibody-protein complex, which is the key to immunoprecipitation. There are usually two options for the type of antibody :

Monoclonal antibody

Highly specific and able to recognize specific epitopes of the target protein.

Polyclonal antibody

It recognizes multiple epitopes of the target protein and has a wide range of recognition.

Capture of Antibody-protein Complexes

In order to separate the antibody-target protein complex, protein A / G agarose beads or magnetic beads are often used as carriers in the experiment. Protein A / G can bind to the Fc region of the antibody, so it can bind to the antibody by binding to the antibody. After adding antibodies, antibody-protein complexes can be captured in the following two ways :

Centrifugation

The antibody-protein complex is separated from the sample by high-speed centrifugation. Particles (antibody-protein complexes) were precipitated at the bottom of the test tube, while other dissolved substances were removed.

Magnetic separation

The antibody-protein complex was captured from the sample using magnetic beads. The magnetic beads are separated from the composite by an external magnetic field.

Washing

In order to remove unbound impurities and non-specifically bound molecules, washing is required. Washing solution (salt-containing buffer) is usually used for multiple washings. This process helps to remove background noise and improve the specificity of the experiment. The composition of the washing solution can be optimized as needed to ensure that excess impurities are removed without affecting the target protein complex.

Elution of Target Protein

After the target protein is successfully enriched, the elution step is followed to separate the target protein from the antibody-complex. Common elution methods include :

Competitive antigen elution

Competitive dissociation of antibody-protein complexes using the same or similar antigens (usually certain peptides of the target protein) as the target protein.

Changing pH Elution

Use a low-pH or high-pH buffer to disrupt the binding between the antibody and the target protein, thereby eluting the target protein.

After elution, the target protein can be analyzed by SDS-PAGE, Western Blot and other techniques.

Analysis and Detection

The target protein after elution can be subjected to a series of analyses. Commonly used methods include :

SDS-PAGE

The protein was separated by electrophoresis according to the molecular weight, and the purity and molecular weight of the protein were analyzed.

Western Blot

Used to detect the presence and expression of specific proteins, and to identify the target protein by labeled antibodies.

Mass spectrometry

For further identification and quantification of the target protein.

Figure 1. Schematic representation of the stages of the immunoprecipitation protocol pre sented in Basic Protocol 1. (1) Cell lysis: antigens are solubilized by extraction of the cells in the presence or absence of detergents. To increase specificity, the cell lysate can be precleared with protein A–agarose beads (steps 15 to 17, not shown). (2) Antibody immobilization: a specific antibody is bound to protein A–agarose beads. (3) Antigen capture: the solubilized antigen is isolated on antibody-conjugated beads. (Reference source: Immunoprecipitation. doi:10.1002/0471140864.ps0908s18）

Application of immunoprecipitation

Protein-protein Interaction Studies

By immunoprecipitation, other proteins that interact with the target protein can be captured.

Enrichment and Analysis of Protein Complexes

Used to analyze the composition and function of protein complexes.

Detection of Antigenic Epitopes

IP technology can identify the binding of antibodies to specific antigens.

Protein Modification Research

For the study of protein phosphorylation, acetylation and other modifications.

Alpha Lifetech has many years of project experience and can better serve the needs of customers. Alpha Lifetech provides immunoprecipitation (IP) services with high specificity and sensitivity to ensure efficient extraction and analysis of target proteins from various biological samples.

FAQ

1. In immunoprecipitation experiments, if the target protein is contaminated by non-specific binding proteins, it will lead to high background signals or low specificity results. The high background signal will affect the analysis of the experimental results and reduce the detectability of the target protein.

Solutions : (1) Optimize antibody selection : Select highly specific monoclonal antibodies or validated antibodies. Ensure that the antibody targets the only epitope of the target protein and avoid cross-reaction with the non-target protein. (2) Use appropriate washing buffer : increase the number of washing steps, and use high-salt buffer to remove non-specific binding molecules. The composition of the washing solution should be optimized according to the characteristics of the antibody and the target protein. (3) Use of blocking agents : Blockers (such as BSA or bovine serum albumin) can be used to block possible non-specific binding sites before antibody binding to reduce the background.
2. Sometimes the experiment can not get the expected target protein precipitation, may be because the target protein is not fully combined with the antibody, or the affinity of the antibody is low, resulting in the target protein is not effectively enriched.

Solutions : (1) Optimize antibody concentration : ensure that the antibody concentration used is appropriate. Too low antibody concentration may lead to insufficient binding, and too high concentration may lead to non-specific binding. (2) Increasing incubation time and temperature : Prolonging the incubation time of the antibody and the sample, usually incubation overnight at 4 ° C can improve the binding efficiency. (3) Use appropriate protein A / G carrier beads : Select suitable carrier beads (such as protein A, protein G, magnetic beads) and ensure that the beads can effectively bind to the antibody. (4) Cell lysis optimization : Ensure that the appropriate cell lysis buffer is used, and optimize the lysis conditions (such as the pH value of the lysis solution, salt concentration, etc.) according to the properties of the target protein.
3. The target protein may be degraded during immunoprecipitation, resulting in the inability to obtain effective precipitation results. This is usually due to the presence of proteases in the sample, or the cleavage conditions are not suitable for stabilizing the protein.

Solutions : (1) Adding protease inhibitors : Protease inhibitors (such as PMSF, EDTA, protease inhibitor mixture, etc.) were added to the lysate to prevent protein degradation. (2) Optimize the lysis conditions : use appropriate lysis buffer to avoid excessive cell disruption or damage to the structure of the target protein. (3) Low temperature operation : Try to keep low temperature operation in all steps to reduce the risk of protein degradation.
4. The precipitated target protein is incomplete, and some target proteins may remain in the supernatant, resulting in a small number of target proteins recovered.

Solutions : (1) Optimize the centrifugation speed and time : ensure that the centrifugation conditions can completely precipitate the antibody-protein complex. For smaller particles, it may be necessary to increase the centrifugation time or increase the centrifugation speed. (2) Using Magnetic Beads : Using magnetic beads can capture antibody-protein complexes more efficiently and reduce losses. (3) Increase the proportion of antibody and carrier beads : appropriately increase the amount of antibody and carrier beads to improve the capture efficiency.
5. In the results of immunoprecipitation, there may be a large number of non-target protein impurities, resulting in the purity of the target protein is not high, affecting the subsequent analysis (such as Western Blot).

Solutions : (1) Multiple washing : Use appropriate washing buffer and perform multiple washings to remove non-specifically bound proteins. The salt concentration of the washing solution can be appropriately increased to help remove some non-specific binding molecules. (2) Different washing schemes are used : sometimes according to the nature of the target protein, the composition of the washing buffer can be changed, such as increasing the concentration of the detergent (such as Triton X-100) to help remove membrane proteins or other unwanted proteins. (3) Select the appropriate antibody and carrier beads : Use antibodies and carrier beads that are suitable for the target protein to ensure that they have a high affinity for the target protein and reduce the interference of impurities.

reference

[1] DeCaprio J, Kohl TO. Immunoprecipitation. Cold Spring Harb Protoc. 2017;2017(12):pdb.prot098640. Published 2017 Dec 1. doi:10.1101/pdb.prot098640

[2] Bonifacino JS, Dell'Angelica EC, Springer TA. Immunoprecipitation. Curr Protoc Protein Sci. 2001;Chapter 9:. doi:10.1002/0471140864.ps0908s18

[3] Wang L, Sanchez J, Hess D, Matthias P. Immunoprecipitation of HDAC6 and Interacting Proteins. Methods Mol Biol. 2023;2589:493-508. doi:10.1007/978-1-0716-2788-4_32

[4] Mohammed H, Taylor C, Brown GD, Papachristou EK, Carroll JS, D'Santos CS. Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for analysis of chromatin complexes. Nat Protoc. 2016;11(2):316-326. doi:10.1038/nprot.2016.020

[5] Sanz LA, Chédin F. High-resolution, strand-specific R-loop mapping via S9.6-based DNA-RNA immunoprecipitation and high-throughput sequencing. Nat Protoc. 2019;14(6):1734-1755. doi:10.1038/s41596-019-0159-1