RPPA is a high-throughput screening tool for proteins. Many different cell line- /tumor sample lysates are spotted with an automated non-contact spotter onto nitrocellulose-coated glass slides. A single slide can contain up to 500 different lysates (plus technical replicate spots). The spotting of up to 50 identical replicate slides per spotting run is possible. The sample consumption is very low, only 10µl lysate are needed for the production of 50-100 replicate slides. A slide is incubated with a specific target-protein antibody. Due to the production of replicate slides, many different antibodies can be applied to the same biological samples. A quality control step, which allows for total protein quantification of the different samples, is performed directly on a replicate slide and enables us for error detection and data normalization. Please find a graphical overview [here].
RPPAs allow for a precise quantification of specific target protein expression and phosphorylation/modification. We use this method in a systems biology approach to quantify pathway signalling of breast cancer cells. A complex network of surface receptors in cancer cells gets stimulated and inhibited by receptor-specific agents. The effects of the signalling modulations are quantified in a time resolved manner on the protein level. RPPA allows for high-throughput screening of many different samples/timepoints in parallel.
Screening of tumour samples:
Quantification of diagnostic markers / quantification of protein kinases activation status of human cancer biopsy material. I mainly worked with Gastrointestinal Stromal Tumors (GIST) in cooperation with Dr. F.Haller / Göttingen. We quantified the expression of more than 50 proteins in about 100 patients. The high number of technical replicates and the use of three biological replicates per patient allows for creating a detailed expression profile.
RNAi knockdown quantification on protein level:
In cooperation with Özgür Sahin we quantified the efficiency of siRNA mediated mRNA knockdown on protein level. Target genes were knocked down in single, double and triple knockdowns in parallel, then the mRNA expression and the protein expression were quantified and compared. This approach was used to disturb a complex signalling network in tamoxifen resistant breast cancer cells (via RNAi). The effects on the cell cycle progression were quantified on the protein level using RPPA.