Reverse phase protein arrays (RPPA) offer great promise for the identification and use of critical biomarkers for drug screening and diagnostics. Samples prepared from tissue lysate that are spotted for RPPA can often produce dilute protein concentrations, sometimes only a few hundred µg/ml. For single spot depositions in an array, this utilizes less than 1% of the available binding capacity for porous nitrocellulose film. The unsurpassed protein binding capacity of porous nitrocellulose (40 µg/cm2) makes it ideally suited for RPPA and detection of low abundance targets in complex protein mixtures, and far exceeds the capacity (approx. 500x) of other protein microarray surfaces (Figure 1).
In order to detect low abundance proteins found in tissue lysates, many investigators rely on signal amplification methods that incur increased reagent costs and can potentially confound data due to amplification of non-specific signal. An alternative approach to signal amplification is to take advantage of the high binding capacity of porous nitrocellulose by performing multiple microarray depositions of dilute samples, and effectively increasing the protein concentration per microarray spot. Coupled with enhanced signal/noise ratios in the red and near IR emission range (due to lower intrinsic fluorescence for nitrocellulose at these wavelengths), this approach can produce very sensitive detection of RPPAs without the use of signal amplification. To illustrate this point, we deposited increasing numbers of repetitions from dilute cell lysates (500 µg/ml) for the detection of P-EGFR (Y1173). Detection was performed using either red or near-IR fluorescence scanning and without the use signal amplification (Figure 2).
Figure 1. Shown are dilution series of purified IgG-TRITC dissolved in a typical Tris/SDS RPPA lysis buffer deposited on various protein microarray slide types. Slides include SuperNOVA porous nitrocellulose, PATH non-porous nitrocellulose, three leading hydrogel providers, and three leading silane-based chemistries (aldehyde, epoxy, amine). Slides were blocked according to the manufacturer’s recommendations and extensively washed prior to detection by fluorescence of the internal TRITC control at 532nm using a GenePix 4000B (Molecular Devices).
(A) Representative scan of an RPPA after detection for p-EGFR (Y1173) with IRDye800 (Li-cor) and scanned using an InnoScanIR (Innopsys). The duplicate lanes marked “NEG” are cell lysates from untreated A431 cells and the two lanes marked “POS” are cell lysates from EGF-treated A431 cells spotted at a 500 µg/ml concentration. Spotting rows proceed from 1 deposition (top) to 10 depositions (bottom) of 400 pL each using a Scienion S3 non-contact printer.
(B) Signal obtained from replicate arrays (n=3) using a GenePix 4400A (Molecular Devices) for detection of Alexa647 and an InnoScanIR (Innopsys) for detection of IRDye800.
(C) Signal-to-Noise obtained with either scanner/dye combination highlighting the improved sensitivity in the near infrared over visible wavelengths when using porous nitrocellulose.