**2. Protein-protein interaction assays**

Protein-protein interaction (PPI) assays can be classified into three broad categories, i.e., in vivo, in vitro, and in silico. (1) The in vivo techniques apply the whole procedure on the living cell or organism itself. (2) In vitro methods require the whole procedure completed outside the cell in a controlled environment in a laboratory, i.e., affinity chromatography, tandem affinity purification (TAP), protein fragment complementation, X-ray crystallography, co-immunoprecipitation, phage display, nuclear magnetic resonance, spectroscopy, and protein arrays. (3) The techniques that are performed by using computers or computer simulations are called in silico techniques. The sequence and structure-based approaches, gene fusion, chromosome proximity, gene expression-based approaches, domain pair-based approach, in silico two hybrid approaches, phylogenetic profile, and phylogenetic tree are some approaches which are based on in silico methods [4].

## **2.1. In vivo techniques for the prediction of protein-protein interactions**

The in vivo technique used to study PPIs is yeast two hybrid (Y2H) method [9]. The two protein domains are involved in the Y2H assay. First domain is the DNA binding domain which helps in binding the DNA and the second one is activation domain that is involved in activation of the transcription of the specific DNA. These two domains are required for the transcription of a particular reporter gene [10]. The interacting proteins that are involved in the Y2H assays must be present in the close vicinity or inside the nucleus because these proteins have the capability to activate reporter gene and the proteins that are not present in nucleus do not have the ability to activate reporter genes. Some other techniques being used are fluorescence resonance energy transfer (FRET), biomolecular fluorescence complementation (BiFC), and bioluminescence resonance energy transfer (BRET) [4].

#### **2.2. In vitro techniques for the prediction of protein-protein interactions**

To learn PPIs in the inherent environment of the cell, a technique called TAP tagging [11] was developed. TAP tagging method was first used to analyze the yeast interactome in a high throughput way [12]. TAP tagging involves two steps, first is double tagging of the protein of interest and second is two-step process of purification [13]. After the process, the proteins that remain attached with the target protein can be studied by using SDS-PAGE and then mass spectrometry analysis is performed to confirm the PPI partner of the protein of interest [14]. TAP tagging used in combination with mass spectrometry which can identify both protein complexes and protein interactions.

The proteins that have many interactions include transcription factors and enzymes [8]. Though, PPIs encompass heterogeneous procedures mostly and the possibility of their regulation is enormous. Various interactions and the outcome of these interactions are needed to identify the better understanding of PPIs inside the cell [3]. By using methods like mass spectrometry, protein chip technology, phage display, and two hybrid system, PPIs data have been increased in recent years [3]. These experimental resources are useful for constructing comprehensive PPI networks. But, day by day the increase in the amount of data on protein-protein interactions is becoming a challenge for validation in the laboratory. To understand the functions of unexplored protein by using computational approaches is necessary

Protein-protein interaction (PPI) assays can be classified into three broad categories, i.e., in vivo, in vitro, and in silico. (1) The in vivo techniques apply the whole procedure on the living cell or organism itself. (2) In vitro methods require the whole procedure completed outside the cell in a controlled environment in a laboratory, i.e., affinity chromatography, tandem affinity purification (TAP), protein fragment complementation, X-ray crystallography, co-immunoprecipitation, phage display, nuclear magnetic resonance, spectroscopy, and protein arrays. (3) The techniques that are performed by using computers or computer simulations are called in silico techniques. The sequence and structure-based approaches, gene fusion, chromosome proximity, gene expression-based approaches, domain pair-based approach, in silico two hybrid approaches, phylogenetic profile, and phylogenetic tree are

The in vivo technique used to study PPIs is yeast two hybrid (Y2H) method [9]. The two protein domains are involved in the Y2H assay. First domain is the DNA binding domain which helps in binding the DNA and the second one is activation domain that is involved in activation of the transcription of the specific DNA. These two domains are required for the transcription of a particular reporter gene [10]. The interacting proteins that are involved in the Y2H assays must be present in the close vicinity or inside the nucleus because these proteins have the capability to activate reporter gene and the proteins that are not present in nucleus do not have the ability to activate reporter genes. Some other techniques being used are fluorescence resonance energy transfer (FRET), biomolecular fluorescence complementa-

To learn PPIs in the inherent environment of the cell, a technique called TAP tagging [11] was developed. TAP tagging method was first used to analyze the yeast interactome in a high

nowadays.

4 Protein-Protein Interaction Assays

**2. Protein-protein interaction assays**

some approaches which are based on in silico methods [4].

**2.1. In vivo techniques for the prediction of protein-protein interactions**

tion (BiFC), and bioluminescence resonance energy transfer (BRET) [4].

**2.2. In vitro techniques for the prediction of protein-protein interactions**

Affinity chromatography is also used to study PPIs in vitro. It is very sensitive technique and can identify even the weakest interactions among the proteins. Though, it generates many of the false positive results because of the great specificity of the proteins. Therefore, studies of protein interactions cannot depend only on affinity chromatography. So, other techniques are needed in combination with affinity chromatography to further confirm the results generated. The affinity chromatography is often combined with mass spectroscopy and SDS-PAGE to produce more convincing results [4].

Co-immunoprecipitation is another in vitro technique that is used for the confirmation of PPIs by using the complete cell extract wherever proteins are present in a complicated blend of cellular machinery and in their natural form that is essential for the significant interactions of proteins [4]. Protein arrays are also being used to study PPIs. A piece of glass is used in which different protein molecules are attached in an organized fashion [15]. Protein microarray analysis gained marvelous importance to do high throughput analysis by running many of the parallel analysis in an automated procedure. The PPIs can be studied by using another proteomics method known as protein fragment complementation assay (PCA). It consists of a family of assays that can be used to identify the proteins of any molecular weight and it provides very simple and straight conducts to determine PPIs in living cells, in vitro, and multicellular organisms [4].

Mass spectroscopy can also be used to determine protein-protein interactions. There are two ways to identify PPIs by using mass spectroscopy shotgun proteomics and peptide finger printing [16]. To analyze complicated mixtures, shotgun proteomics is the most suitable technique while in the peptide finger printing, SDS-PAGE is used to separate the eluted complex. X-ray crystallography can also be used to determine PPIs in vitro. It is a type of microscopy with very high resolution that is used for the identification of proteins at atomic level and it is helpful for functional analysis of proteins [17]. The analysis of PPIs can also be done by using nuclear magnetic resonance (NMR) spectroscopy. In NMR spectroscopy, the magnetically active nuclei that are surrounded by a strong magnetic field engross electromagnetic radiations at distinguishing frequencies that are monitored by the chemical surroundings [18].

#### **2.3. In silico techniques to predict protein-protein interactions**

Many of the in vivo and in vitro techniques generate a large amount of data that is helpful in the development of software and tools for the identification of PPIs among various proteins that are found in many different combinations. The computational methods used for the in silico prediction of interactions among proteins may include the tools described in **Table 1**.


**Author details**

Faisalabad, Pakistan

University Press; 2009

10.1155/2014/147648

DOI: 10.1016/S1570-0232(02)00074-0

627. DOI: 10.1038/35001009

**References**

, Mahmood-ur-Rahman Ansari1,2\* and Muhammad Iqbal3

3 Department of Environmental Science and Engineering, GC University-Faisalabad,

1 Department of Bioinformatics and Biotechnology, GC University-Faisalabad, Faisalabad,

Introductory Chapter: Protein-Protein Interactions and Assays

http://dx.doi.org/10.5772/intechopen.77337

7

[1] Braun P, Gingras AC. History of protein-protein interactions: From egg-white to complex networks. Proteomics. 2012;**12**(10):1478-1498. DOI: 10.1002/pmic.201100563

[2] Nooren IMA, Thornton JM. Diversity of protein-protein interactions. The EMBO Journal.

[3] Zhang A.Protein Interaction Networks: Computational Analysis. NewYork, NY: Cambridge

[4] Rao VS, Srinivas K, Sujini GN, Kumar GNS. Protein-protein interaction detection: Methods and analysis. International Journal of Proteomics. 2014;**2014**(2):1-12. DOI:

[5] Yanagida M. Functional proteomics; current achievements. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. 2002;**771**(1-2):89-106.

[6] Berggård T, Linse S, James P. Methods for the detection and analysis of protein-protein

[7] Phizicky EM, Fields S. Protein-protein interactions: Methods for detection and analysis. Microbiological Reviews. 1995;**59**(1):94-123. DOI: 10.1111/j.1471-4159.2009.06024.x

[8] Sarmady M, Dampier W, Tozeren A. HIV protein sequence hotspots for crosstalk with

[9] Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, Knight JR, Lockshon D, Narayan V, Srinivasan M, Pochart P, Qureshi-Emili A, Li Y, Godwin B, Conover D, Kalbfleisch T, Vijayadamodar G, Yang M, Johnston M, Fields S, Rothberg JMA. Comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature. 2000;**403**(6770):623-

interactions. Proteomics. 2007;**7**(16):2833-2842. DOI: 10.1002/pmic.200700131

host hub proteins. PLoS One. 2011;**6**(8). DOI: 10.1371/journal.pone.0023293

\*Address all correspondence to: mahmoodansari@gcuf.edu.pk

2003;**22**(14):3486-3492. DOI: 10.1093/emboj/cdg359

2 Central Hi-Tech Lab, GC University-Faisalabad, Faisalabad, Pakistan

Munazza Ijaz<sup>1</sup>

Pakistan

**Table 1.** The list of in silico tools and their links to predict protein – protein interactions.
