A SYSTEMS APPROACH TO QUANTITATIVE MULTIPLEXED RPPA’S
Presented by: Michael A. Shultz, PhD
RPPA Workshop in Kobe, Japan
Porous nitrocellulose film (PNC) is the substrate of choice for RPPA largely because of its extraordinarily high protein binding capacity, which can exceed non-porous planar counterparts by 500X. Spotting, assay, and analyte preservation reagents developed specifically for PNC-based microarrays contribute needed sensitivity and accuracy to RPPA assays. An additional property of PNC, which we call Resonance Scattering Amplification (RSA), when properly employed can increase dynamic range of RPPA by two orders of magnitude. Fluorescent has advantages over colorimetric signal development for quantitative RPPA applications. PNC autofluorescence, relatively high at visible wavelengths, is non-existent at NIR wavelengths, so that assay backgrounds are equivalent to clear glass. In RPPA, quantum nanocrystals have advantages over organic fluorophores, which are related to brightness and stability. For multiplexing, quantum nanocrystals have added advantages related to narrow emission bands with minimum spectral overlap. By combining the RSA property of PNC with PNC-specific reagents and NIR-emitting quantum nanocrystals, we were able to achieve amole-zmole assay sensitivities which allowed us to multiplex antibodies using an inexpensive high-speed imaging instrument (ArrayCAMTM). Using this system, we are able to discriminate phosphorylated from non-phosphorylated Akt not just in the same tissue lysate, but in the same arrayed spot. This level of multiplexing facilitates the internal standardization parameters necessary for quantitative comparisons within signal pathways.