Reverse Phase Protein Array (RPPA)
RPPA is a fluorescent antibody-based proteomic technology that can quantify multiple proteins in hundreds of samples with 10 µL of lysates in a high-throughput fashion. It involves depositing nL of lysates onto a nitrocellulose or PVDF membrane bound to a microslide as 100-200 µm spots using a microarray printing instrument such as the Marathon Classic System from Arrayjet available at CESB. The membrane on the slide can be divided into multiple areas (up to 64), each reacting with a specific antibody, followed by fluorescent secondary antibody and imaging using a microarray scanner such as the InnoScan 710 outfitted with an auto slide loader from Innopsys housed in CESB. The microspot intensity can be converted to protein concentration by calibrating against authentic protein standards. RPPA offers much more accurate quantification of proteins than Western blotting as no electrophoretic transfer is needed and a dilution series is used to check response linearity. It does require validation of antibody specificity. Thanks to the popularity of ELISA testing, the inventory of validated antibody is rapidly expanding. CESB has purchased many metabolism and immune function-related antibodies, and will continue to expand this list.