Quantitative Multiplex Immunoprecipitation
Quantitative Multiplex Immunoprecipitation - Related Videos
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Multiplexed Quantitative Bacterial Detection using Dye-Labeled Magnetic Microspheres
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Source:Rhea Kang1, Gabby Mora1, Sherry Dunbar11Luminex, A DiaSorin Company, Austin.This video demonstrates multiplexed quantitative bacterial detection employing carboxylated magnetic polystyrene microspheres dyed into distinct sets. The procedure encompasses the formation of bead-bacteria complexes, magnetic separation, and flow-based analysis, enabling precise and simultaneous quantification of multiple bacteria in the same reaction well.
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Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation
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Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.
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Methylated DNA Immunoprecipitation
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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).
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Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging
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Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.
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...
FAQs
Two measurements are required: bead identity specifies which bait was captured, while fluorescent intensity indicates how much prey signal was associated with that bait. Because these signals are recorded together, QMI can distinguish interaction profiles for several bait proteins in the same sample. This pairing is central to comparing complex composition rather than merely detecting whether a protein is present.
Each antibody serves a distinct analytical role. An antibody coupled to a bead set selects its corresponding bait from the lysate, whereas a fluorescent antibody reports a prey that remains associated after capture. Keeping these functions separate lets the assay encode bait identity through the bead and interaction-associated signal through fluorescence, supporting parallel comparison of multiple complexes.
By measuring several bait-prey relationships in one multiplexed analysis, QMI can reduce the amount of cell lysate needed compared with running separate immunoprecipitation assays for each interaction. That economy is especially useful when samples are limited or when many conditions must be compared. The resulting dataset emphasizes relative differences in complex composition across samples rather than a single isolated interaction.
Changes in fluorescence associated with a particular bait-prey pair can reflect altered complex composition between samples exposed to different stimuli or experimental states. In the provided use cases, these states include stimulation, mutation, drug treatment, and disease-related conditions. Comparing the corresponding multiplex profiles helps identify which interactions change together rather than treating all proteins as equally affected.
An analysis begins with cell lysates and distinct bead sets carrying antibodies against selected bait proteins. After capture, fluorescent antibodies detect co-immunoprecipitated prey proteins. The bead set identifies the bait during readout, while fluorescence intensity supplies the interaction-associated measurement. Researchers then measure both features by flow cytometry or a multiplex analyzer, allowing the profiles to be compared across samples.
In biochemistry, QMI is suited to experiments that ask how protein-complex composition differs among defined sample states. It can be applied when researchers want to examine signaling networks systematically, assess the effects of a mutation or drug treatment, or compare disease-related conditions. Its multiplex format links several interaction measurements within the same experimental comparison, aiding network-level interpretation.