A method for the covalent capture and screening of diverse small molecules in a microarray format.
This protocol describes a robust method for the covalent capture of small molecules with diverse reactive functional groups in microarray format, and outlines a procedure for probing small-molecule microarrays (SMMs) with proteins of interest. A vapor-catalyzed, isocyanate-mediated surface immobilization scheme is used to attach bioactive small molecules, natural products and small molecules derived from diversity-oriented synthesis pathways. Additionally, an optimized methodology for screening SMMs with purified proteins and cellular lysates is described. Finally, a suggested model for data analysis that is compatible with commercially available software is provided. These procedures enable a platform capability for discovering novel interactions with potential applications to immunoglobulin profiling, comparative analysis of cellular states and ligand discovery. With the appropriate materials and experimental setup, the printing of SMMs can be completed in 14 hours over 3 days. Screening and data analysis requires 2 days. A detailed timeline is provided.
Bradner
James E
JE
Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA.
McPherson
Olivia M
OM
Koehler
Angela N
AN
D002384:Catalysis; D017953:Isocyanates; D008024:Ligands; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding
D017953:Isocyanates; D008024:Ligands