The Application of the Chemical Array for Biological Study
The identification of specific interactions between small molecules and human proteins of interest is a fundamental step in chemical biology and drug development. The small molecules that bind to specific proteins can be used as tools to study the functio
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Introduction Chemical arrays represent one of the promising and highthroughput approaches to search ligands against proteins of interest. Successful applications of chemical arrays to the discovery of small molecule ligands for a variety of proteins have been reported (1–4). One of the key steps in the technology is immobilization of small molecules on glass slides (5–8). We have developed a nonselective immobilization method that allows various compounds to be immobilized on an array slide in a functional Mahesh Uttamchandani and Shao Q. Yao (eds.), Small Molecule Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 669, DOI 10.1007/978-1-60761-845-4_8, © Springer Science+Business Media, LLC 2010
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group-independent manner (9). Previous screening using the glass slides where small molecules were immobilized by this immobilization method remained a limitation because it required purified protein (10). Bradner and coworkers reported that mammalian cell lysates that overexpressed target protein could be used for binding assays using chemical arrays (11). We have adopted this system and modified it to create a ligand screening method that enables us to detect the binding ligands of human proteins of interest with a high-throughput manner (12). The success of screening using chemical arrays depends on the choice of target proteins and a number of small molecules to be immobilized on array slides (13). A chemical library, NPDepo (RIKEN Natural Products Depository), was established by our laboratory (14). However, the number of proteins that could be used for chemical array screening was still limited. Hence, we have constructed a human gene library, GLORIA, to accelerate screening on our platform. Our screening method can perform large-scale chemical array screening using the gene library, GLORIA, and a chemical library, NPDepo.
2. Materials 2.1. Cloning of Human cDNAs and Establishment of GLORIA
1. pGEM-T-Easy vector (Promega, Madison, WI). 2. pDsRed-Express-N1 vector (Clontech, Mountain View, CA). pDsRed-Express-N1 is encoded DsRed-Express. DsRedExpress, a variant of Discosoma sp. red fluorescent protein (DsRed), forms a homotetramer. This DsRed protein is designated as RFP in this report. 3. Automated sequencer (Applied Biosystems, Foster City, CA).
2.2. Preparation of Mammalian Cell Lysates that Overexpress RFP-fused Proteins
1. RPMI 1640 medium (Invitrogen, Tokyo, Japan) supplemented with 10% fetal calf serum (FCS; Nichirei Biosciences, Tokyo, Japan). 2. Effectene Transfection Reagent (QIAGEN, Tokyo, Japan). 3. Phosphate-buffered saline (PBS): Prepare 10× stock with 1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH2PO4. Prepare 1× solution before using it. 4. Amount of protein in each lysate was measured with the Bio-Rad protein Assay Reagent (BioRad, Hercules, CA). 5. TyphoonTM 9400 imager (GE Healthcare, Tokyo, Japan). 6. 4× sampling buffer (250 mmol/L Tris-HCL (pH 6.8), 40% glycerol, 8% SDS, 20% 2-mercaptoethanol, and 0.04% bromophenol blue).
Chemi
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