Protein–protein interaction studies on protein arrays: Effect of detection strategies on signal-to-background ratios

Hurst, Robin; Hook, Brad; Slater, Michael; Hartnett, Jim; Storts, Douglas R.; Nath, Nidhi

doi:10.1016/j.ab.2009.05.028

Cited by 38 publications

(29 citation statements)

References 40 publications

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“…This simplifies the process, and significantly improves the uniformity of the arrays and reproducibility of the assays. The rapid preparation of HaloTag-fusion arrays and excellent performance opens-up the possibility for screening hundreds or thousands of different protein interactions [56, 57]. …”

Section: Key Applications Of Halotag Technologymentioning

confidence: 99%

HaloTag, a Platform Technology for Protein Analysis

Urh

Rosenberg²

2013

CCG

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Understanding protein function and interaction is central to the elucidation of biological processes. Systematic analysis of protein interactions have shown that the eukaryotic proteome is highly interconnected and that biological function frequently depends on the orchestrated action of many proteins. Perturbation of these functions or interactions can lead to various disease states and pharmacologic intervention can result in corrective therapies. The fact that proteins rarely act in isolation, but rather comprise complex machines that stably and/or transiently interact with many different partners at different times, demands the need for robust tools that allow comprehensive global analyses of these events. Here we describe a powerful protein fusion technology, the HaloTag platform, and how it enables the study of many facets of protein biology by offering a broad choice of applications. We review the development of the key aspects of the technology and it’s performance in both in vitro and in vivo applications. In particular, we focus on HaloTag’s multifunctional utility in protein imaging, protein isolation and display, and in the study of protein complexes and interactions. We demonstrate it’s potential to help elucidate important facets of proteomic biology across complex biological systems at the biochemical, cell-based and whole animal level.

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Section: Key Applications Of Halotag Technologymentioning

confidence: 99%

HaloTag, a Platform Technology for Protein Analysis

Urh

Rosenberg²

2013

CCG

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“…Independent methods are needed to validate and discover protein-protein interactions [12]. We have used the HaloTag technology in a number of formats as a means of identifying or validating a number of binary protein interactions and also to identify constituents of multi-protein complexes [7, 40]. …”

Section: Use Of Halotag Recombinant Proteins To Identify Protein-protmentioning

confidence: 99%

“…We have used the HaloTag technology for a number of functional studies, including protein microarrays, affinity purification of DNA-protein, protein-protein interactions, and protein complex identification [7, 17]. The HaloTag is a modified haloalkane dehalogenase designed to covalently bind a series of chloroalkane derivatives such as fluorophore-labeled ligands (Promega).…”

Section: Introductionmentioning

confidence: 99%

The HaloTag: Improving Soluble Expression and Applications in Protein Functional Analysis

Peterson

Kwon²

2013

CCG

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Technological and methodological advances have been critical for the rapidly evolving field of proteomics. The development of fusion tag systems is essential for purification and analysis of recombinant proteins. The HaloTag is a 34 KDa monomeric protein derived from a bacterial haloalkane dehalogenase. The majority of fusion tags in use today utilize a reversible binding interaction with a specific ligand. The HaloTag system is unique in that it forms a covalent linkage to its chloroalkane ligand. This linkage permits attachment of the HaloTag to a variety of functional reporters, which can be used to label and immobilize recombinant proteins. The success rate for HaloTag expression of soluble proteins is very high and comparable to maltose binding protein (MBP) tag. Furthermore, cleavage of the HaloTag does not result in protein insolubility that often is observed with the MBP tag. In the present report, we describe applications of the HaloTag system in our ongoing investigation of protein-protein interactions of the Y. pestis Type 3 secretion system on a custom protein microarray. We also describe the utilization of affinity purification/mass spectroscopy (AP/MS) to evaluate the utility of the Halo Tag system to characterize DNA binding activity and protein specificity.

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“…Cell-free based protein microarrays have been applied to immunological studies [25], vaccine development [26,27], early detection of biomarkers [27,28], biochemical activity [21] protein-protein interaction studies [10,28], such as protein-protein, protein-DNA, protein-RNA, protein-phospholipids, and protein-small molecule interactions [22] and toxin detection [29,30]. Over the last few years, several in situ expressed microarrays have been developed such as: Protein in situ arrays (PISA), printing protein arrays from DNA (DAPA) arrays and Nucleic Acids Programmable Protein Arrays (NAPPA) [9,11,17].…”

Section: Concept Of Protein Microarrays and Current Applicationsmentioning

confidence: 99%

Data Analysis Strategies for Protein Microarrays

et al. 2012

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Microarrays constitute a new platform which allows the discovery and characterization of proteins. According to different features, such as content, surface or detection system, there are many types of protein microarrays which can be applied for the identification of disease biomarkers and the characterization of protein expression patterns. However, the analysis and interpretation of the amount of information generated by microarrays remain a challenge. Further data analysis strategies are essential to obtain representative and reproducible results. Therefore, the experimental design is key, since the number of samples and dyes, among others aspects, would define the appropriate analysis method to be used. In this sense, several algorithms have been proposed so far to overcome analytical difficulties derived from fluorescence overlapping and/or background noise. Each kind of microarray is developed to fulfill a specific purpose. Therefore, the selection of appropriate analytical and data analysis strategies is crucial to achieve successful biological conclusions. In the present review, we focus on current algorithms and main strategies for data interpretation.

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Protein–protein interaction studies on protein arrays: Effect of detection strategies on signal-to-background ratios

Cited by 38 publications

References 40 publications

HaloTag, a Platform Technology for Protein Analysis

HaloTag, a Platform Technology for Protein Analysis

The HaloTag: Improving Soluble Expression and Applications in Protein Functional Analysis

Data Analysis Strategies for Protein Microarrays

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