3.2. Antibody Validation
Reliability of RPPA results largely depends on the quality of the antibodies as signals cannot be differentiated in “specific” and “unspecific” like for conventional immunoblotting techniques. Unfortunately, commercial available antibodies often do not meet the demand necessary for RPPA. For antibody validation Schuster et al. propose a step-by-step procedure combining immunohistochemistry and immunoblot analysis from FFPE-tissues using the same antibody. This kind of tandem validation identifies antibodies that bind specific to the respective antigen at the right molecular weight (Western Blot) and allows identification of antibodies that can localize the protein of interest in tissue sections (IHC). By combining Western Blot and IHC antibody validation could be improved [56]. Alternatively, protein extracts of several different cell lines (ten or more) can be a starting point for validation. The first criterion for a specific antibody is a single band at the correct molecular weight for protein extracts of cell lines obtained by Western Blot (Figure 3). The antibody may also be considered specific, if several protein bands appear that correspond to isoforms, cleavage products, dimer formation or mutations (i.e., “explainable bands” as for example for ERK1/2 or p95 and p185 in the case of HER2). Often, antibodies are tested against stimulated and unstimulated cell lines in order to detect phosphorylated proteins [57,58,59]. After analysis of cell lysates, the next step for antibody validation is Western Blot analysis using protein lysates of the material which will be used for RPPA later on, e.g., frozen or FFPE‑tissue. This step is crucial because antigen detection in cell lines might deviate from results obtained in tissues samples [56]. In a further test, quantification results obtained by RPPA are compared with those of Western Blot. If the results are comparable, e.g., stronger staining in IHC and higher protein amounts in the same cases by RPPA analysis, the antibody is suitable for RPPA studies. Figure 4 summarizes the steps for antibody validation.
Figure 3  Antibody validation by Western Blot using different cell lines. The left panel shows an antibody that is suitable for RPPA analysis as it detects one single protein band at the expected molecular weight. The antibody used for the right panel should not be used for RPPA studies as there are numerous bands besides the proposed “specific” band. 1–4: lysates from breast cancer FFPE-tissue; 5–8: lysates from different cell lines; arrows indicate the expected molecular weight.   To facilitate antibody validation the RPPA community is making an international effort to create an upcoming website with detailed information of antibody validation protocols and already validated antibodies [10]. Already available antibodies cover a broad range of pathways, including proliferation, apoptosis, angiogenesis, and epithelial-mesenchymal-transition. An antibody list for RPPA studies can be found for example at the MD Anderson RPPA website [60]. The number of specific and high affinity antibodies targeting the epitope of interest is still limited. Especially the number of phosphor‑specific and other post-translational modification specific antibodies is low [4].
Figure 4  Basic steps for antibody validation. For detailed explanations of the different steps see text. WB, Western Blot.

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