Protoarray human protein microarray v5.0 protein protein interaction kit

CTX used for the protein array analysis was chemically synthesized using Fmoc chemistry with 2-chlorotrityl resin and oxidized using 0.1 M ammonium bicarbonate pH 8 and 1 mM reduced glutathione, with stirring overnight at room temperature. The peptide was biotinylated using a 20-fold excess of EZ-Link® Sulfo-NHS-LC-biotin (ThermoFisher Scientific), according to the manufacturer’s instructions. The biotinylated sample was analyzed utilizing an SCIEX 5800 MALDI TOF/TOF mass spectrometer (SCIEX, Foster city, CA, USA), followed by purification of labeled CTX (to remove excess non-reacted and hydrolyzed biotin reagent from the solution) via HPLC. The purified sample was dried and resuspended in 100 μl of 1.0% PBS (pH 7.2) to give a final concentration of 10 μg. Protein-protein interactions among biotinylated CTX and more than 9,000 human proteins were then investigated employing a ProtoArray® Human Protein Microarray v5.0 Protein-Protein Interaction (PPI) kit (Invitrogen™) and significant interactions detected employing a GenePix® microarray scanner (Molecular Devices), according to the manufacturer’s instructions.

Biotinylation of PA₃₆₆ for protein interaction analysis was achieved utilising a 20-fold excess of EZ-Link^® Sulfo-NHS-LC-biotin (ThermoFisher Scientific, Waltham, MA, USA), and following the manufacturer’s instructions. Successful labelling of PA₃₆₆ was confirmed by molecular weight analysis using a SCIEX TOF/TOF™ 5800 mass spectrometer (SCIEX, Framingham, MA, USA), followed by RP-HPLC purification of labelled PA₃₆₆ to remove excess non-reacted and hydrolyzed biotin reagent from the solution. The purified sample was dried and resuspended in 100 µL of 1.0% PBS (pH 7.2) to achieve a required final amount of 10 µg. Interactions between biotinylated PA₃₆₆ and more than 9000 human proteins were investigated employing a ProtoArray^® Human Protein Microarray v5.0 Protein-Protein Interaction (PPI) kit (Invitrogen™, Carlsbad, CA, USA), and significant interactions detected using a GenePix^® 4000Bmicroarray scanner (Molecular Devices, Sunnyvale, CA, USA), according to the manufacturer’s instructions.