Retrogenix cell microarray screen

Screening for PfTRAP binding proteins was performed using the Retrogenix Cell Microarray technology. Initially 4336 expression vectors, each encoding a unique human plasma membrane protein and the ZsGreen1 reporter constructs, were pre-spotted on glass slides using a QArray mini microarrayer (Genetix, UK). Human HEK293 cells were grown over the arrays and reverse-transfected, resulting in the over-expression of each membrane protein and ZsGreen1. Slides were then incubated with QDot ITK Carboxyl Quantum Dots, 585 nM (Life Technologies cat # Q21311MP) which were pre-coupled with Ni–NTA and coated with His-tagged PfTRAP, giving a final PfTRAP concentration of 100 nM. PfTRAP binding proteins were identified by fluorescence imaging (at ~ 500 nm wavelength for ZsGreen1 and at ~ 585 nm for QDots) using an Ettan DIGE imager (GE Healthcare) and analysed using ImageQuant software (GE). For confirmation and specificity testing, expression vectors encoding the PfTRAP binding proteins were re-spotted on new slides, and HEK293 cells were reverse-transfected. Slides were then treated with the same Ni–NTA-coupled QDots coated with His-tagged PfTRAP as before, or with His-tagged PDL1 (each at 100 nM final concentration), or with no ligand conjugated. Interactions were analysed by fluorescence imaging using an Ettan DIGE Imager.