Definition
The pull-down assay is an in-vitro technique used to detect physical interactions between two or more proteins and an invaluable tool for confirming a predicted protein-protein interaction or identifying novel interacting partners.
Purpose
Protein interactions reveal a lot about how proteins and cells function under different conditions. A pull-down assay allows us to look at direct protein interactions and utilizes a bait protein bound to beads in a column to catch protein binding partners.
This technique can be used to verify a predicted protein interaction via Western blot or identify novel protein interactions using a total protein stain.
Pull-down assays can be used to identify novel protein interactions or assess how proteins bind under different conditions for any number of diseases, or simply provide information that can offer a better picture of what’s happening in the inner world of cells.
1. Affinity pull-down assay
Definition
Pull-down assays involve isolation of a protein complex by adsorbing the complex onto beads. Immobilized ligands on the beads bind specifically to a component of the complex, either via an affinity tag (e.g., GST, histidine, maltose binding protein, etc.) or an antibody.
Pull-down assays are important tools for mapping protein-protein interaction networks. They have been successfully used on a global scale to map protein-protein interactions in a number of organisms (e.g., yeast, E. coli, C. elegans).
Process
Design of the appropriate controls for the experiment are essential.
Affinity pull-down assays are well established for the isolation and subsequent identification of protein complexes. GST pull-down assays involve affinity purifications of one or several unknown proteins from a biological sample using a GST-tagged bait protein.
The basic principle is that the GST-tagged bait protein binds to its partners, and the resulting complex is captured on beads with immobilized glutathione. A control is included to identify false positives that bind to GST in the absence of a bait protein.
The control can either be lysate from separately transformed cells that express GST (not the bait fusion protein) or lysate from non-transformed cells to which GST is added.
2. Tandem affinity purification
Definition
Tandem Affinity Purification, (or “The TAP method”) with or without modifications, has been used to purify and identify complexes from yeast, trypanosomes, fruit flies, humans, and plants. TAP employs two successive affinity chromatography steps to isolate protein complexes. The rationale behind the use of two affinity steps is to enhance the specificity of the purification procedure and hence reduce the number of false positives. Furthermore, the conditions used throughout this method are mild -- in order to preserve complex integrity and to maximize yield. The method has been used successfully in many cases.
Process
A bait protein (a known or suspected complex component) is tagged with both protein A and calmodulin binding peptide (CBP) with a tobacco etch virus (TEV) protease cleavage site between the two tags.
The double-tagged bait protein is expressed at near natural levels, since overexpression may trigger non-physiological interactions. The complex is first adsorbed to IgG Sepharose beads, via the protein A tag, and eluted by cleavage with TEV.
3.Identifying Protein Interactions via Co-immunoprecipitation
Definition
Immunoprecipitation is a well-established technique that uses antibodies to isolate antigens from crude biological samples.
The name is historical and originates from analysis methods based on the precipitation reaction obtained when mixing antibody and antigen at the correct ratio.
Overview
Co-immunoprecipitation uses antibodies directed towards one (known or supposed) component of a complex. The antibody binds to its antigen, which is part of a multiprotein complex. The antibody-protein complex assembly is then captured through the addition of protein A or protein G Sepharose beads to the mixture.