Definition for View source for Pull-down assay

The pull-down assay is an in vitro method used to determine a physical interaction between two or more proteins. Pull-down assays are useful for both confirming the existence of a protein-protein interaction predicted by other research techniques (e.g., co-immunoprecipitation) and as an initial screening assay for identifying previously unknown protein-protein interactions. 

Pull-down assays are a form of affinity purification and are very similar to immunoprecipitation, except that a "bait" protein is used instead of an antibody. Affinity chromatography (i.e., affinity purification) methodologies greatly enhance the speed and efficiency of protein purification and simultaneously provide the technology platform to perform a pull-down, or co-purification, of potential binding partners. In a pull-down assay, a bait protein is tagged and captured on an immobilized affinity ligand specific for the tag, thereby generating a "secondary affinity support"’ for purifying other proteins that interact with the bait protein. The secondary affinity support of immobilized bait is then incubated with a protein source that contains putative "prey" proteins, such as a cell lysate. The source of prey protein at this step depends on whether the researcher is confirming a previously suspected protein-protein interaction or identifying an unknown interaction. The method of protein elution depends on the affinity ligand and ranges from using competitive analytes to low pH or reducing buffers. 

Besides investigating the interaction of two or more proteins, pull-down assays are a powerful tool to detect the activation status of specific proteins. For example, proteins that are activated in response to tyrosine phosphorylation can be pulled down using an immobilized SH2 domain that targets the phosphorylated tyrosine on a given protein. Additionally, GTPases, which act as molecular switches that regulate cell signaling by cycling between a GTP-bound (active) and GDP-bound (inactive) state, can be pulled down using an immobilized GTPase-binding domain of downstream proteins that are recruited to GTP-bound, activated GTPases. In both types of pull-down assays, because the specificity of the interaction is dependent on the sequence of the binding domain, these approaches are highly specific in detecting the activation of distinct proteins.<br> 

[[Image:Pull-down-shematic-734px.jpg]]<br> 

'''General schematic of a pull-down assay.''' A pull-down assay is a small-scale affinity purification technique similar to immunoprecipitation, except that the antibody is replaced by some other affinity system. In this case, the affinity system consists of a glutathione S-transferase (GST)-, polyHis- or streptavidin-tagged protein or binding domain that is captured by glutathione-, metal chelate (cobalt or nickel)- or biotin-coated agarose beads, respectively. The immobilized fusion-tagged protein acts as the "bait" to capture a putative binding partner (i.e., the "prey"). In a typical pull-down assay, the immobilized bait protein is incubated with a cell lysate, and after the prescribed washing steps, the compexes are selectively eluted using competitive analytes or low pH or reducing buffers for in-gel or Western blot analysis.<br>

'''The Pull-Down Assay as a Confirmatory Tool'''

The confirmation of previously suspected interactions typically utilizes a prey protein source that has been expressed in an artificial protein expression system. This allows the researcher to work with a larger quantity of the protein than is typically available under endogenous expression conditions and eliminates confusing results that could arise from interaction of the bait with other interacting proteins present in the endogenous system that are not under study. Protein expression system lysates (i.e., E. coli or baculovirus-infected insect cells), in vitro transcription/translation reactions, and previously purified proteins are appropriate prey protein sources for confirmatory studies.

'''The Pull-Down Assay as a Discovery Tool'''

The discovery of unknown interactions contrasts with confirmatory studies because the research interest lies in discovering new proteins in the endogenous environment that interact with a given bait protein. The endogenous environment can entail a plethora of possible protein sources but is generally characterized as a complex protein mixture considered to be the native environment of the bait protein. Any cellular lysate in which the bait is normally expressed, or complex biological fluid (i.e. blood, intestinal secretions, etc.) where the bait would be functional, is an appropriate prey protein source for discovery studies.<br>

'''Pull-Down Methodologies'''

Homemade pull-down approaches for confirming or identifying protein-protein interactions are ubiquitous in contemporary scientific literature. The homemade pull-down assay represents a collection of reagents from multiple commercial vendors that cannot be validated together as a functional assembly except by extensive assay development by the researcher, and troubleshooting this combination of reagents can be tedious and time-consuming. Commercial pull-down kits contain complete, validated sets of reagents specifically developed for performing pull-down assays. The buffers provided in each kit allow complete flexibility to determine the optimal conditions for isolating interacting proteins. The working solutions for washing and binding are physiologic in pH and ionic strength, providing a starting point from which specific buffer conditions for each unique interacting pair can be optimized. Many commercial kits also incorporate spin columns for efficient handling of small volumes of affinity support, complete retention of the affinity support during the pull-down assay and thorough washing of the protein complexes for minimal nonspecific protein pull-down, all of which are common sources of variability and high background using traditional pull-down assay formats.<br>