Proteomics reverse phase protein array data

For the correction of anti-species secondary antibody staining, arrays were assayed in the absence of primary antibodies. For the measurement of spotted protein, one blank chip (i.e., without antibody incubation) was stained with SYPROÒRuby. Scanned images were analyzed using ZeptoVIEW 3.1 software (Bayer Technology Services GmbH). Normalized fluorescence intensities (NFIs) for each sample and protein target were calculated as reference fluorescence intensities of primary antibody stained arrays (RFIprimary) corrected for secondary antibody staining (RFIsecondary), as well as relative spotted protein concentration (RFIprotein), determined by (RFIprimary − RFIsecondary) / RFIprotein, using ZeptoVIEW 3.1 software. NFI values were used for subsequent analysis.
A series of bovine serum albumin references were spotted on each array. Each sample underwent serial dilution (100%, 75%, 50%, and 25%) to assess linearity. Prior to RPA analysis, all antibodies were validated by western blotting to assess their specificity.