Figure 3: Detection of proteins generated in situ from individual samples.
From: Personalised proteome analysis by means of protein microarrays made from individual patient samples
(a) Quality assessment of on-chip PCR by oligonucleotide hybridisation; two oligonucleotides were used, labelled red or green, respectively, each binding to one of the DNA-strands. Typical results are shown with an oligonucleotide specific to only one PCR-product present in quadruplicate (left) and a simultaneous hybridisation with oligonucleotides to all PCR-products (right). (b) Fusion of the images obtained after an incubation with fluorescently labelled antibodies against N- (red signal) and C-terminal tags (green signal) of the expressed seven tumour marker proteins. Spots 8 and 9 were negative controls without DNA-template. (c, d) Protein detection with labelled antibodies that target proteins CDK2 and TP53, respectively. (e) Results obtained on arrays produced from tissue samples of individual patients. Normal = healthy pancreas; CP = chronic pancreatitis; PDAC = pancreatic ductal adenocarcinoma; MiaPaca-2 = PDAC cell line. All proteins were identified with a tag-specific antibody (left). Binding patterns obtained with two different, isoform-specific antibodies. One isoform of the RUNX1 protein was present in all samples (right); the other one was found in diseased material only (middle). (f) Ninety-six DARPin binders were expressed in situ, each in three copies. Tag-specific antibodies identified all binders (left). The other two panels (middle, right) show binding patterns obtained upon incubation with protein AKT3. The white frames indicate the 16 binders that were expected to interact with AKT3. Different washing stringency produced distinct variations in the binding patterns.
