Protein-protein interactions (PPIs) are the basis of many important cellular processes such as signal transduction, molecular transport and various metabolism pathways, while aberrant PPIs are the basis of multiple aggregation-related diseases, such as Alzheimer’s disease, and may lead to cancer. Therefore, PPIs have been studied extensively in the area of bioscience and medical research. Here, we review the major contemporary approaches utilized for detection and analysis of protein protein interaction.
Table1: Summary of common PPI detection methods
|An immunoprecipitation experiment designed to affinity-purify a bait protein antigen together with its binding partner using a specific antibody against the bait.|
|Pull-down Assays||An affinity chromatography method that involves using a tagged or labeled bait to create a specific affinity matrix that will enable binding and purification of a prey protein from a lysate sample or other protein-containing mixture.|
|Yeast two-hybrid (Y2H)||Monitor complex formation through transcriptional activation of reporter genes.|
|Far-Western Blotting||Similar strategy to Western blotting with one key difference. The antibody probe in a typical Western blotting detection is substituted with an labeled bait protein as the probe.|
|Tandem affinity purification-mass
|TAP-MS is based on the double tagging of the protein of interest on its chromosomal locus, followed by a two-step purification process and mass spectroscopic analysis.|
|Protein microarrays||Microarray-based analysis allows the simultaneous analysis of thousands of parameters within a single experiment.|
|Bio-Layer Interferometry(BLI)||Change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time.|
|Surface Plasmon Resonance (SPR)||SPR angle changes with surface refractive indexes, which is in direct proportion to the molecular mass of the biomolecule attached to the metal surface.|
Co-immunoprecipitation (Co-IP) is a popular technique to identify and validate physiologically relevant protein-protein interactions. By using target protein-specific antibodies to indirectly capture proteins that are bound to a specific target protein, Co-IP is applied to screening novel protein-protein interactions or confirming the existence of protein-protein interactions.
In Co-IP proteins interact in a non-denaturing condition which is almost physiological. However, low affinity or transient interaction between proteins may not be detected. On the other hand, the result of Co-IP could not determine whether the interaction is direct or indirect, since the possibility of involvement of additional proteins could not be ruled out.
Pull-down assay is an in vitro method used to determine a physical interaction between two or more proteins. It can be used for confirmation of existing protein-protein interactions discovered by other techniques or initial screening to identify novel protein-protein interactions.
The basic principle of pull down assay is to utilize a tag fused protein (such as GST-tag, His-tag and biotin-tag) immobilized to affinity resin as the bait protein. Proteins binding to the bait protein (prey protein) can be captured and “pulled down” when the target protein or cell lysate flows through. By subsequent elution and analysis using Western Blot or Mass Spectrometry, a predicted interaction can be confirmed or previously unknown interactions can be discovered.
The two-hybrid system is one of the most widely used methods to screen or confirm protein–protein interactions. Two protein domains are required in the Y2H assay which will have two specific functions: (i) a DNA bindingdomain (DBD) that helps binding to DNA, and (ii) an activation domain (AD) responsible for activating transcription of DNA. Both domains are required for the transcription of areporter gene. Y2H analysis allows the direct recognition of PPI between protein pairs. However, the method may incur a large number of false positive interactions. On the other hand, many true interactions may not be traced using Y2H assay, leading to false negative results.
Far-western blotting is a molecular biological method which is based on the technique of western blotting to detect protein-protein interaction in vitro. While usual western blotting uses an antibody to detect a protein of interest, far-western blotting uses a non-antibody protein, which can bind the protein of interest. Thus, whereas western blotting is used for the detection of certain proteins, far-western blotting is rather employed to detect protein protein interactions.
TAP tagging was developed to study PPIs under the intrinsic conditions of the cell. This method is based on the double tagging of the protein of interest on its chromosomal locus, followed by a two-step purification process. Proteins that remain associated with the target protein can then be examined and identified through SDS-PAGE followed by mass spectrometry analysis, thereby identifying the PPI collaborator of the original protein of interest.
Protein microarrays are rapidly becoming established as a powerful means to detect proteins, monitor their expression levels, and probe protein interactions and functions. A protein microarray is a piece of glass on which various molecules of protein have been affixed at separate locations in an ordered manner. Protein microarrays have seen tremendous progress and interest at the moment and have become one of the active areas emerging in biotechnology.The objective behind protein microarray development is to achieve efficient and sensitive high-throughput protein analysis, carrying out large numbers of determinations in parallel by automated process.
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