Advanced Technologies for Studies on Protein Interactomes: Protein Microarray, Protein Chips, Yeast Two-Hybrid System, Mass Spectrometry and cDNA Library Screening
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cDNA Library Screening, Protein Microarray, Yeast Two-Hybrid System, Mass Spectrometry
Abstract
Assigning functions to the numerous genes uncovered by large-scale sequencing projects is a major obstacle for modern biology, as only a small portion of gene activity can be deduced directly from the coding sequence. Understanding the function of proteins relies heavily on identifying their interactions with one another. Direct detection of intermolecular interactions through protein microarray, yeast two-hybrid system, mass spectrometry fluorescent techniques to visualise protein complexes or pull-down assays, and technologies detecting functional interactions between genes, such as RNAi knock down or functional screening of cDNA libraries, are the main technologies used to investigate protein-protein interactions and assign functions to proteins. Significant progress has been achieved in the aforementioned methods in the past few years. Here we take a look back at recent advances and how they've changed gene function annotation. Several ProteinChip arrays with chemically modified surfaces (pre-activated) that form covalent bonds with free amine groups are available from Bio-Rad Laboratories (Hercules, CA). They are developed for diverse biological applications including biomarker discovery, protein profiling, protein–protein interaction studies, peptide mapping for protein ID, immunoassays, and receptor-ligand binding studies. These arrays have eight 2-mm diameter spots (A–H format) that correlate with the spacing of wells in a single column of a standard 96-well microplate and are therefore amenable for high-throughput applications in varied robotics. Here, we describe two types of arrays that are particularly amenable to covalently immobilize biomolecules for subsequent capture of targeted proteins from complex biological samples. The arrays differ in their surface chemistry and hence both should be tested to determine the most suitable array for the application envisioned. Other considerations include lower non-specific binding and higher sensitivity.
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