Reverse phase protein arrays (RPPA) offer great promise for the identification and analysis of critical biomarkers for disease states in patient samples and for high-throughput drug screening using cultured cell systems. Porous nitrocellulose (NC) film slides continue to be the preferred substrate for RPPA due to their high protein binding capacity and many functional advantages.
A necessary component of an RPPA experiment is to accurately define the protein content of arrayed sample lysates. A convenient method is to perform an on-slide quantification post-arraying. Data presented here highlight a simple method for quantifying total lysate protein spotted on RPPA arrays by using SYPRO® Ruby protein stain with ONCYTE Film Slides.
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Figure 1. SYPRO® Ruby Protein Quantification on ONCYTE Film Slides.
Figure 1. Total protein may be quantified after spotting on ONCYTE Film Slides by staining with SYPRO® Ruby. Shown is a range of standard protein (BSA) at spotting concentrations ranging from 0.01 – 5 mg/ml after on-slide staining with SYPRO® Ruby. Fluorescence was quantified using two common excitation wavelengths (532 and 635 nm) and was obtained with an Axon 4000B microarray scanner.
Figure 2. Fluorescent Signal from SYPRO® Ruby Stained Protein on ONCYTE Film Slides.
Figure 2. Fluorescent signal after staining with SYPRO® Ruby is linear over 3 orders of magnitude. Shown is the dynamic range of protein staining with SYPRO® Ruby on a SuperNOVA nitrocellulose film slide blocked with Super G blocking reagent. BSA standard was spotted in various RPPA lysate buffers with R2 averaging 0.9955 for all of the buffers (Buffer A: SDS/Tris, Buffer B: SDS/Tris/Triton; Buffer C: Urea; Buffer D: Grace Bio-Labs RPPA lysis buffer).
