One of the main methods for detection of interaction between two proteins is yeast two-hybrid system or Y2H (Berggård, et al, 2007). This method is based on the fact that many transcription factors are composed of two domains, DNA binding domain (BD) and Activator domain (AD), which are not covalently bound together (Phizicky and Fields, 1995). In this system we need to make two plasmids i)composed of BD and a trap ii) composed of AD and our target protein. Two plasmids are then inserted to a cell to let them co-expressed (Berggård, et al, 2007). In case of interaction between trap and target protein, two domains are brought together and transcription will be induced. (Dang, C. V. et al, 1991)
Figure 1. Two hybrid system. Right: Target and Trap match together. Left: Target and Trap do not match together.
Another method for detection of protein-protein interaction is Biotinylation which is similar to Y2H. In this method one of the proteins is fused to biotin ligase and the other protein is attached to the substrate of this enzyme. If our proteins of interest interact with each other, the enzyme will catalyzes the binding of biotin to the specific site of substrate (Fernandez-Suarez, et al, 2008).
Tandem Affinity Purification (TAP)-tag
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This method is a two-step affinity chromatography which its combination with Mass spectrometry can be used for detection of interacting proteins with a target protein. In this method two different tags with a protease sensitive linker between them are added to Bait (Berggård, et al, 2007). For this purpose genes encoding these tags, linker peptide and Bait protein are linked together as shown in figure 2. First tag corresponds to protein A which binds IgG in chromatography beads and the other is a Calmodulin binding peptide (CBP). (Rigaut et al. 1999)
Figure2. mechanism of Tandem Affinity Purification
As shown in figure 2, the engineered gene is introduced to yeast for expression. In the next step cells are lysed to put the extract in affinity chromathography. In the first chromathography, IgG antibodies are connected to beads which bind to Protein A. by adding a protease, the linker protein degrades and first tag dissosiate from comlex. In the second round of chromathography, beads are attached to Calmodulin which interacts with CBP. The aim of second step is removing protease and other contaminants of the first chromathography step. Elution by EDTA release our complex. (Rigaut et al. 1999)
This methods are based on inducing the fluorescence resonance energy transfer . FRET is a form of energy transference phenomenon that occurs between two fluorescent molecules with less than 5-6 nm distance in space (Barnard et al. 2007) and (Tsien. 1998). In Fluoresence-based techniques, two proteins of interest are labled by two different fluorescent probes. One of this kind of probes is Green Fluorescent Protein or GFP. When target proteins interact with each other the two fluorescent proteins brought closer together . As a result, the spectrum emitted by one of them can overlap the excitation spectrum of the other and FRET accures. This technique can be used as a tool for monitoring of protein-protein interactions in vivo under a fluorescence microscope (Barnard et al. 2007).
Figure 3. Mechanism of fluorescence-based techniques. Left: there is no interaction between proteins and therefore FRET doesn't occur. Right: the proteins interact together and bring two fluorescent molecules closer so FRET occurs.
Immunoprecipitation is a kind of classical purification procedure that can determine if two different proteins interact. In this method Antibody against the known protein is added to cell lysis. All of proteins in cell extract interacting with target protein will be purified as a binding complex to antibody (see figure 4). Western blot can be used for identification of proteins in complex. (Phizicky and Fields, 1995).
Figure 4. Immunoprecipitation method for identification of protein-protein interactions
All of the mentioned methods as well as other techniques for identification of interaction between proteins have strong and weak points which some of them are mentioned in table 1.
Table 1. Comparison of different methods for detection of protein-protein interactions
Always on Time
Marked to Standard
-Simple to set up
-Transient and week interactions easily detected
- little information regarding interaction localization
-large number of false positive
-Berggård, et al, 2007
-good spatial resolution in cell
-Fernandez-Suarez, et al, 2008
Tandem Affinity Purification
-Highly sensitive and selective
-little information regarding interaction localization
- Transient and week interactions may be lost during series of purification
- low yield
-Berggård, et al, 2007
-can report on the dynamics and localization of the interactions within a cell(1)
-limited sensitivity and dynamic range(2)
- Risk of false-negatives if the two fluorescent tags are positioned beyond the Förster distance(3)
-1. Prasher, 1995
-2. Fernandez-Suarez, et al, 2008
-3. Barnard et al. 2007
-Small amount of sample
-provide information on specificity of interaction, affinity and rate of association or disassociation (1)
- Immobilized protein may be inactivated
-studying interactions close to the surface may result in parameters that are not true in solution (2)
-1. Jason-Moller et al, 2006
-2. Berggård, et al, 2007