Immunoprecipitation: Applications, Uses, & Types

Immunoprecipitation: Applications, Uses, & Types

is a small-scale affinity purification technique to enrich or isolate a protein from biological samples, such as tissue or cell lysate. It works on the principle of antigen-antibody interaction.

Specific antibodies are immobilized on solid support made of agarose resin or magnetic beads. A solution containing antigen is added to the matrix which leads to complex formation between the specific protein and antibodies. The protein complexes are then eluted using an elution buffer, giving purified antigen or protein.

Immunoprecipitation is an extensively used technique in a range of Life Sciences and epigenetics labs to isolate proteins of interest and other biomolecules. It’s useful for subsequent studies of isolated proteins, such as their structure, expression, identity, and post-translational modification.

Different types of immunoprecipitation are practiced in labs based on the goal of the study. For example, a protein-protein interaction study uses the co-immunoprecipitation (Co-IP) technique. Whereas, chromatin immunoprecipitation or RNA immunoprecipitation techniques are used to study protein-nucleic acid interaction.

This article will cover the principle and mechanism of the immunoprecipitation technique, its four types, and its uses in lab workflows.

Need new or refurbished lab equipment? Excedr leases.

See our equipment list and browse a sample selection of what we can source. Or, if you’re ready, request an estimate.

How Does Immunoprecipitation (IP) Work?

Immunoprecipitation works on the principle of the high affinity of an antibody towards the target protein. The specific antibody is incubated with the cell lysate containing the target antigen. This leads to an antibody-protein complex formation.

The complex is added to a matrix made of Protein A or G coupled to magnetic, agarose, or sepharose beads. The beads provide solid support for the immobilization of the complex. Proteins A and G have a high affinity for immunoglobulin (primarily IgG). Hence, they bind to the antibody complex in their Fc region.

At the end of each incubation step, where the solution containing antigen-protein complex and beads are together present in a slurry, centrifugation is performed to remove the solution as supernatant. After the supernatant is removed, a wash buffer is used to wash off unbound complexes, antibodies, or any other contaminants.

Then, the complex is eluted from the bead matrix using an elution buffer for further analysis. The complexes and antigens of interest are either analyzed alone by SDS-PAGE or followed by staining, Western Blotting, or Mass spectrometry protocols.

Figure: An overview of the Immunoprecipitation (IP) experiment.

Factors Affecting Immunoprecipitation (IP) Procedure

There is a range of factors that can impact the output or data accuracy of your immunoprecipitation (IP) experiment:

  • Cell Lysate Preparation: The first step to immunoprecipitation requires the right lysis buffer. It stabilizes native protein conformation, minimizes antibody binding site denaturation, inhibits enzymatic activity, and increases protein release from cells.

Denaturing buffers containing NP-40 and Triton X-100 are routinely used to prepare cell lysate. Further, phosphatase/protease inhibitors should always be added to the lysis buffer to prevent dephosphorylation and proteolysis of IP samples.

  • Antibody selection: The correct primary antibodies are essential to capture target proteins. Polyclonal antibodies (bind multiple epitopes on target protein) form tighter complexes than monoclonal antibodies (bind single epitope on target protein). Moreover, there are fewer chances of antibodies washing off in the washing step.
  • Cell Lysate Preclearing: Likely, non-specific binding to IP antibodies, Protein A/G, or beaded support will occur in lysates, as they are complex mixtures of lipids, proteins, carbohydrates, and nucleic acids. This may adversely affect the detection of immunoprecipitated targets.

Therefore, pre-clearing potentially reactive components from a lysate before the IP experiment is an effective way to prevent the non-specific binding of these components to IP beads.

Types of Immunoprecipitation (IP)

Immunoprecipitation is used to isolate target proteins and study their identity, expression, structure, and modification. Its variations are used to study the interaction of the target protein with other proteins or nucleic acids.

Individual protein immunoprecipitation (IP)

The method is used to isolate a specific protein from a solution containing many different proteins by using an antibody specific to the target protein. Purified proteins are verified by assays like Western blot or ELISA.

Figure: Steps to Individual Protein Immunoprecipitation.

Protein complex immunoprecipitation (Co-IP)

The assay is also known as a “pull-down” assay. It’s used to study interaction partners to the protein of interest, such as receptors, ligands, and co-factors. A specific antibody for a known protein, that is a part of a large complex, is used to pull the entire protein complex out of the solution. Thus, helping the identification and further studies of the unknown components of the complex.

Figure: Steps to Co-Immunoprecipitation.

RNP immunoprecipitation (RIP)

Used to precipitate RNA-binding proteins (ribonucleoproteins). The RNA-protein complex is isolated by RNA extraction and identified by using RT-PCR and cDNA sequencing assays.

A variation to the RIP is photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP).

Figure: Steps to RNA-Immunoprecipitation procedure.

Chromatin immunoprecipitation (ChIP)

It’s used to identify regions of the genome that associate with DNA-binding proteins, such as histones and transcription factors, or to identify specific proteins associated with specific regions of the genome. The isolated DNA is further analyzed by using techniques like PCR, microarrays, sequencing, or some other methods.

Figure: Steps to the Chromatin immunoprecipitation (ChIP) protocol.

What is Immunoprecipitation (IP) Used For?

Immunoprecipitation can be used for a variety of in vivo or in vitro experiments, such as immunoassay, ELISA, immunoblotting, and tagged-protein (having short peptide or fluorescent protein tags for easy visualization of target proteins) pull-down assays.

Protein Purification

In molecular biology labs, Co-IP is an important tool for the purification or enrichment of a target protein, from cell lysate or any biological sample, using a bait protein-specific antibody. The technique is used to assist in a spectrum of studies, such as:

  • Studying protein-protein interactions
  • Identifying the presence and quantity of a protein
  • Detecting enzymatic activity of proteins
  • Studying the rate of synthesis or degradation of the target protein
  • Determining the molecular weight of a polypeptide
  • Detecting post-translational modification

Figure: An overview of protein purification using Immunoprecipitation.

Don’t have the budget to purchase lab equipment outright? Consider leasing through Excedr to save your lab time and money. Browse your leasing options today!

Get the R&D Equipment You Need with Excedr

Immunoprecipitation is a technique used in labs to isolate and enrich target proteins using the antigen-antibody interaction principle. Based on the target molecule to be studied, the technique is of different types” Co-IP, CHIP, tagged proteins, and RIP.

In conjunction with the techniques like western blotting, PCR, and ELISA, immunoprecipitation is used to purify, detect, and measure proteins’ presence and concentration in a sample, study interaction partners, and determine enzymatic activities of target proteins.

High-quality reagents are required to perform IP experiments. It includes lysis buffer, washes buffer, magnetic or agarose beads crosslinking to protein A/G, elution buffer, sample buffer, primary and secondary antibodies, PBS, tris HCL, enzymes, and many others.

However, quality reagents alone aren’t enough to obtain quality, accurate, and validated data from the experiment. You also need cutting-edge instruments to perform such high-throughput techniques.

You might find it challenging to acquire advanced high-tech equipment because of the associated cost, time, and legwork involved in the process. This all comes easy when you choose to lease with Excedr.

It has a comprehensive leasing solution that allows labs and industries like yours to perform their lab operations at ease by offering all desired equipment on lease. You don’t need to pay expensive upfront costs, spend extra on repair and maintenance of the equipment, or waste your time visiting store-to-store for affordable equipment for your lab.

At Excedr, we first understand your requirements, analyze your problem, and then help you choose the equipment that will best suit your lab. The categories of equipment available in our store include analytical equipment, clinical equipment, life sciences and biotech equipment, and many more.

Excedr’s leasing solution is an effective way to run your lab on budget and extend your cash runway for other essential operations. Additionally, it helps you save time to invest in your R&D, grow your lab, and achieve your milestones faster.