Immunoprecipitation Assay
Immunoprecipitation Assay - Related Videos
08:08
Co-Immunoprecipitation and Pull-Down Assays
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2023
Co-immunoprecipitation (CoIP) and pull-down assays are closely related methods to identify stable protein-protein interactions. These methods are related to immunoprecipitation, a method for separating a target protein bound to an antibody from unbound proteins. In CoIP, an antibody-bound protein is itself bound to another protein that does not bind with the antibody, this is followed by a separation process that preserves the protein-protein complex. The difference in pull-down assays is that...
07:13
Immunoprecipitation Assay to Study Enzyme Receptor Interactions in Transfected Cells
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2025
In this video, we demonstrate a technique to perform the co-immunoprecipitation of GFP-tagged wild-type and mutated PD-1 proteins using beads coated with anti-GFP antibodies. The bead-bound PD-1-GFP proteins are incubated with SHP2 proteins that interact with PD-1, enabling the identification of specific structural motifs in PD-1 that play a critical role in SHP2 interaction.
08:39
Chromatin Immunoprecipitation
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2023
Histones are proteins that help organize DNA in eukaryotic nuclei by serving as “scaffolds” around which DNA can be wrapped, forming a complex called “chromatin”. These proteins can be modified through the addition of chemical groups, and these changes affect gene expression. Researchers use a technique called chromatin immunoprecipitation (ChIP) to better understand which DNA regions associate with specific histone modifications or other gene regulatory proteins. Antibodies are used to isolate...
12:36
Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo
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Cited by 22 •
2016
Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.
21:24
Methylated DNA Immunoprecipitation
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Cited by 43 •
2009
This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).
FAQs
The antibody provides the assay’s selectivity by recognizing the target protein within a complex biological sample. This binding determines which target molecules enter the antibody–protein complex, while associated proteins may also be recovered if they remain connected to the target. Consequently, antibody choice directly shapes whether the experiment emphasizes target abundance or protein–protein associations.
Protein A or protein G beads capture the antibody–protein complexes, allowing the bound material to be separated from the rest of the sample. Their role is therefore physical rather than analytical: they provide a recoverable support for the complexes. After capture, washing reduces nonspecific molecules, improving the relevance of the material eventually eluted for analysis.
Washing removes molecules that associate nonspecifically with the antibody–protein complexes, helping distinguish meaningful recovery from background material. Elution then releases the captured contents for downstream examination. The recovered sample can support conclusions about protein abundance or associated partners, but those conclusions depend on whether the experiment is evaluated by Western blotting or mass spectrometry.
A typical workflow begins with a complex biological sample and a specific antibody, followed by formation of antibody–protein complexes. Protein A or protein G beads capture these complexes, and washing removes nonspecific molecules. The retained material is then eluted and examined, commonly by Western blotting to assess protein abundance or by mass spectrometry to identify associated proteins.
Western blotting is useful when the experiment focuses on measuring the abundance of a selected recovered protein. Mass spectrometry is suited to identifying proteins present in the eluted material, including potential interaction partners. Thus, the downstream analysis determines whether the assay primarily addresses a defined protein measurement or a broader survey of associated proteins.
In biology, researchers apply the assay to examine protein expression, post-translational modifications, signaling pathways, and protein–protein interactions. Isolating a selected protein from a complex sample concentrates material relevant to that biological question. Analysis of the eluate can then reveal the target’s abundance or indicate which proteins are associated with it in the studied system.