Pull Down Assay
Pull Down 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...
09:34
Flyback Converter
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2023
Source: Ali Bazzi, Department of Electrical Engineering, University of Connecticut, Storrs, CT. A flyback converter is a buck-boost converter, which can both buck and boost. It has electrical isolation between the input and the output using a coupled inductor or a "flyback transformer." This coupled inductor enables a turns ratio that provides both voltage step-up and step-down capability, like in a regular transformer but with energy storage using the air-gap of the coupled inductor. The...
12:18
DC/DC Boost Converter
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2023
Source: Ali Bazzi, Department of Electrical Engineering, University of Connecticut, Storrs, CT. Boost converters provide a versatile solution to stepping up DC voltages in many applications where a DC voltage needs to be increased without the need to convert it to AC, using a transformer, and then rectifying the transformer output. Boost converters are step-up converters that use an inductor as an energy storage device that supports the output with additional energy in addition to the DC input...
08:16
In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays
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Cited by 3 •
2021
This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro.
10:26
DC/DC Buck Converter
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2023
Source: Ali Bazzi, Department of Electrical Engineering, University of Connecticut, Storrs, CT. While it is simple to step up or down AC voltages and currents using transformers, stepping up or down DC voltages and currents in an efficient and regulated manner requires switching power converters. The DC/DC buck converter chops the input DC voltage using a series input switch, and the chopped voltage is filtered through the L-C low-pass filter to extract the average output voltage. The diode...
FAQs
The affinity tag provides a selective handle that allows the bait protein to associate with affinity beads. Once immobilized, the bait can retain binding partners during incubation with purified proteins or a cell lysate. This arrangement separates bait-associated molecules from unbound material and creates a basis for analyzing whether particular prey proteins are recovered with the bait.
Washing helps remove molecules that associate with the beads or bait nonspecifically, while preserving partners that bind specifically. Controls provide the comparison needed to recognize background capture rather than genuine interaction. Without these steps, proteins detected after elution could reflect nonspecific binding, making conclusions about binding specificity or protein-complex composition less reliable.
Purified proteins allow a pull-down assay to examine a defined binding relationship with fewer molecular components present. A cell lysate provides a broader mixture of cellular proteins, so the assay can reveal associated partners within a more complex biological sample. These formats therefore support different questions about direct binding, binding specificity, or protein-complex composition.
A typical workflow begins by immobilizing the tagged bait on affinity beads, followed by incubation with a cell lysate or purified proteins. Selective washing removes unbound or weakly associated material, and elution releases the retained molecules for analysis. The recovered sample can then be examined to determine which prey proteins or other binding partners accompanied the bait.
SDS-PAGE separates recovered proteins by their migration properties and can reveal the overall pattern of captured material. Immunoblotting tests for specific prey proteins when the target is known. Mass spectrometry provides a way to identify associated proteins more broadly. Selecting among these readouts depends on whether the goal is visualization, targeted detection, or partner discovery.
This approach is useful for investigating signaling pathways, protein complexes, and the specificity of molecular binding. Researchers can compare recovered partners under different experimental conditions to examine changes in associations. The resulting data help connect a biochemical interaction with broader biological mechanisms, especially when identifying which proteins associate with a tagged bait in a cellular context.