3.3. Spotting
The nitrocellulose-coated slides we are using have a size of 7 cm × 2 cm. For RPPA studies only minimal amounts of protein lysates (1 nL of a 2 ng/nL solution) are needed. The samples in our setting are spotted in 3.75 grids (Figure 5). Each complete grid (grids #1–3 in Figure 5) is comprised of 16 subgrids. The smaller grid (grid #4 in Figure 5) is comprised of 12 subgrids. One subgrid consists of three samples in duplicates, most commonly spotted in a five spot two-fold dilution series in duplicates. The dilution series is either performed manually or using an automated liquid handling system (e.g., epMotion from Eppendorf, Hamburg, Germany) in 384-well plates before the spotting process. The dilution series also contains a negative control consisting of protein extraction buffer. On one slide 180 samples can be spotted, including positive and negative controls. For each sample 12 spots (5 fold dilution series, negative control, all in duplicates) are generated. Thus, in total, 2160 spots (12 × 180) are spotted on each slide. Usually proteins are immobilized on nitrocellulose-coated glass using solid pin‑based contact printing, although other printing technologies (piezoelectric or and inkjet spotting) and substrates (e.g., macroporous silicon) have been described [10,61]. Beyond that, the Zeptosens RPPA platform that is based on Planar Wave Guide (PWG) technology permits highly sensitive quantitative protein profiling. Automated systems that are commonly used for RPPA spotting are ArrayIt SpotBot Extreme Microarray Spotter [62] (Arrayit, Sunnyvale, CA, USA), Aushon BioSystems 2470 Microarrayer [19] (Billerica, MA, USA), or SpotArray Microarray printing system [63] (Perkin Elmer, Waltham, MA, USA). Additionally, control samples should also be included in at least duplicates to allow optimal readout and quality control. The spotting devise and other factors, such as temperature and humidity, can affect the spot size. Therefore temperature and humidity should be controlled during the whole spotting process. In our setting a humidity of 60%–65% and a temperature of 14–15 °C to avoid precipitation of SDS, limiting sample evaporation, and optimal sample zone diameter works very well. Other spotting devices or extraction buffer compositions may require different conditions.
Figure 5  RPPA-spotting pattern detected by chemiluminescent detection. Subgrids comprise 6 samples, each in a five-spot serial dilution plus protein extraction buffer as negative control (36 spots). Grids comprise 16 subgrids and 576 spots (Grid 1–3)/12 subgrids and 432 spots (Grid 4). In total, 180 samples can be spotted per slide (2160 spots). Numbers (1–3) indicate three samples spotted in duplicates. * Smaller grid (3/4 size of Grids 1–3).

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