PMC:4996393 / 21506-23623
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4996393","sourcedb":"PMC","sourceid":"4996393","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4996393","text":"3.4. Signal Detection\nThe immobilized proteins are detected with antibodies whose specificity for the antigen of interest has been validated as described above. The presence of antigen-antibody binding can be detected via near-infrared dyes, chromogenic reporter, chemiluminscence, or planar waveguide technologies [10]. The generated signal is proportional to the primary and, indirectly, to the secondary antibody bound to the spotted proteins, and may be quantified to estimate relative protein concentration. Near-infrared fluorescence dyes are often used [64]. Advantage of fluorescence detection is its large dynamic range; however, photo bleaching and quenching might cause false decreases in the total signal. Chromogenic detection is possible with a multitude of colorless chemical substrates that form a colored product when substrate and enzyme are interacting [65]. This signal detection method is compatible with the use of automated staining stations often used for immunohistochemistry, such as the DAKO autostainer or similar devices, allowing high-throughput staining of RPPA slides. Protein detection by horseradish peroxidase (HRP) that produced light when acting on chemiluminescent substrates (e.g., ECL solution) is shown in Figure 5. In the case of Zeptosens RPPA platform specific analysis software is needed, which is provided by the supplier. For the mentioned RPPA detection methods, it could be shown that it is possible to detect proteins in the fg/mL range with linearity in the pg/mL range [66]. In our hands, chemiluminescent signal detection was the most sensitive and flexible method, allowing even the detection of very low abundant proteins, i.e., transcription factors. However, the following points need to be kept in mind when using chemiluminescence: Although signal detection is very flexible, it is time dependent and, therefore, the comparison of signal intensities from different studies can be a challenge. Bridging samples are one solution to solve this problem. Furthermore, the resolution of the images may be suboptimal which may compromise the quality of the results.","divisions":[{"label":"Title","span":{"begin":0,"end":21}}],"tracks":[{"project":"2_test","denotations":[{"id":"27600215-24777629-69479537","span":{"begin":316,"end":318},"obj":"24777629"},{"id":"27600215-17309101-69479538","span":{"begin":561,"end":563},"obj":"17309101"}],"attributes":[{"subj":"27600215-24777629-69479537","pred":"source","obj":"2_test"},{"subj":"27600215-17309101-69479538","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec9593","default":true}]}]}}