Name: protein capture with antibodies
Uploaded: 2023-04-30T11:57:59.000Z
Duration: 1 min 20 s
Channel: JoVE

Immunoprecipitation is used to isolate a single protein from a complex mixture, such as a cell extract.
It employs antibodies specific against the target protein. These antibodies are immobilized on magnetic or agarose beads by direct covalent linking or via antibody-binding recombinant bacterial proteins, such as protein A or G.
When incubated with the protein sample, the pre-coated beads allow the antibodies to form a complex with the target proteins.
Next, the sample is subjected to low-speed centrifugation to precipitate the antibody-protein complex.
The pellet is then resuspended in a mild buffer with low pH or high salt conditions. This breaks the protein-antibody bonds and releases the target protein into the solution.
Finally, the suspension is again centrifuged at a low speed to obtain the target protein in the supernatant.
Like immunoprecipitation, co-immunoprecipitation uses the same principle to study protein-protein interactions.
In this case, a member protein from a protein complex is used as bait to separate its other binding partners.

immunoprecipitation

Immunoprecipitation

Immunoprecipitation, or IP, is a widely used technique that employs protein-antibody interactions to isolate proteins or protein complexes in their native state for studying protein-protein interactions, quaternary structures, or supramolecular complexes. Various modifications of the technique, including chromatin IP, cross-linking IP, and fluorescence IP, are commonly used.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation, also known as ChIP, is used to study protein-DNA or protein-RNA interactions. It is an important technique for studying crucial cellular processes such as gene transcription, DNA replication, recombination and repair, cell cycle progression, and epigenetics. It is also helpful to identify the in vivo location of binding sites of various transcription factors, histones, and other regulatory proteins.
The major steps in ChIP include fixation, sonication, immunoprecipitation, and analysis. It involves cross-linking the target protein with the DNA using a fixing agent, such as formaldehyde, followed by sonication or enzymatic hydrolysis to obtain smaller chromatin fragments. The technique then utilizes high-affinity antibodies specific against the protein of interest to capture the DNA bound to the protein in an immunoprecipitation reaction. The cross-linking is then reversed using high heat, high salt concentration, and proteinase K to release the DNA from associated proteins. The DNA is further purified to prepare it for analysis.
Cross-linking Immunoprecipitation
Cross-linking immunoprecipitation, or CLIP, identifies the regions of protein binding sites on endogenous RNA by co-precipitating them in the active transcription phase. RNA molecules are cross-linked to proteins to hold them together tightly and prevent their degradation. The procedure for the breakdown and isolation of the complexes is similar to ChIP. This technique is used to study the interaction of RNA with RNA binding proteins and their modifications in various biological systems.
Limitations of IP
Though immunoprecipitation techniques tend to reduce the number of purification steps, it has certain limitations. The antibody binding recombinant bacterial proteins, proteins A or G, conjugated to agarose beads, and antibodies used in the method may undergo non-specific binding, introducing contaminants in the purified protein preparation. Also, the immobilization of antibodies on beads requires optimization and is time-consuming. The washing of the beads after the antigen-antibody complex is critical, but there is a chance of losing the target protein at this step.

Immunoprecipitation, or IP, is a widely used technique that employs protein-antibody interactions to isolate proteins or protein complexes in their native ...

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JoVE Core

Cell Biology

Unit 1

Analyzing Cells and Proteins

KEY TERMS AND CONCEPTS

protein capture with antibodies

Immunoprecipitation uses antibodies to capture a specific protein or protein complex from a sample. The protein stays in its native state, which helps scientists study protein-protein interactions, quaternary structure, and larger supramolecular complexes.

Several related methods build on this idea. Chromatin immunoprecipitation, or ChIP, is used to study protein-DNA and protein-RNA interactions. Cross-linking immunoprecipitation, or CLIP, is used to find where proteins bind endogenous RNA during active transcription.

ChIP is important for studying gene transcription, DNA replication, recombination and repair, cell cycle progression, and epigenetics. It also helps identify the in vivo binding sites of transcription factors, histones, and other regulatory proteins. The main steps are fixation, sonication, immunoprecipitation, and analysis.

In ChIP, a fixing agent such as formaldehyde cross-links the target protein to DNA. The sample is then broken into smaller chromatin fragments by sonication or enzymatic hydrolysis. High-affinity antibodies against the protein of interest capture the DNA-protein complex, and the cross-links are later reversed with high heat, high salt, and proteinase K. The DNA is then purified for analysis.

CLIP follows a similar breakdown and isolation workflow. RNA is cross-linked to proteins so the complex stays together and is less likely to degrade. This method is used to study RNA interactions with RNA-binding proteins and their modifications in different biological systems.

Immunoprecipitation also has limits. Antibodies, agarose beads linked with protein A or G, and bacterial recombinant proteins can show non-specific binding and add contaminants. Antibody immobilization on beads needs careful optimization and can take time. Washing the beads is critical, but the target protein can be lost at this step.

Protein Capture with Antibodies

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Immunoprecipitation uses antibodies to capture a specific protein or protein complex from a sample. The protein stays in its native state, which helps scientists study protein-protein interactions, quaternary structure, and larger supramolecular complexes. Several related methods build on this idea. Chromatin immunoprecipitation, or ChIP, is used to study protein-DNA and protein-RNA interactions. Cross-linking immunoprecipitation, or CLIP, is used to find where proteins bind endogenous RNA...

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key terms and concepts

Learn how antibodies capture proteins in immunoprecipitation, plus how ChIP and CLIP reveal protein-DNA and protein-RNA interactions.

Protein Capture with Antibodies | JoVE K12

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JoVE Core Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

Protein Capture with Antibodies

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