Abstract:
Proteins play a pivotal role in cellular processes. As proteins represent the major ‘workforce’ of biomolecules, changes in protein abundances and in posttranslational modifications indicate changes in the behavior of cells on the molecular level. Analysis of proteins improves our understanding of physiological and pathophysiological mechanisms. Today, there are numerous technologies and methods available for protein analysis. For semi-quantitative detection, Western-Blotting still represents the gold standard and is probably the most widespread method for protein analysis.
The DigiWest approach which is described in this thesis is based on the Western-Blot. Transfer onto a bead-based microarray platform creates hundreds of replicas of an initial blot. Proteins are separated by SDS-PAGE and blotted onto a membrane as performed for a classical Western-Blot. The membrane containing the size-separated proteins is then cut into molecular weight fractions. Proteins are eluted from the molecular weight fractions and each fraction is loaded onto a distinct color-coded Luminex bead set. The color code of the bead sets retains the molecular weight information and allows pooling of all bead sets loaded with proteins into a bead-mix which represents the initial Western-Blot with a resolution of 0.5 mm. As only a small aliquot of a bead-mix is required for an antibody-incubation, a bead-mix loaded with protein from a single Western-Blot is sufficient for hundreds of antibody incubations which are performed in microtiter plates. After fluorescence-based readout on the Luminex instrument, the color code of the beads allows digital reconstruction of the initial Western-Blot lanes using the antibody specific signal obtained on the different bead-sets representing molecular weight fractions of the initial blot. Therefore, the method was named ‘DigiWest’. The digital data allow fast quantification of the antibody specific signal without image processing and the data can also be used to create gray-scale images mimicking classical Western-Blot images. Although hundreds of replicas are generated, allowing hundreds of antibody incubations in fast and automatable assays, DigiWest uses the same sample amount that is usually used for a single Western-Blot while it keeps sensitivity and signal linearity comparable to high-end Western-Blot readout platforms. The results are robust and highly comparable to classical Western-Blots. Application of the DigiWest method allowed a comprehensive analysis of laser capture microdissected samples which are barely sufficient for a single Western-Blot.
Mouse models play an important role in toxicology and the liver, the most important organ for drug metabolism, gains special attention. Besides its homogenous appearance the liver is built from lobules as repeated units and zonation results in different metabolic competences for the hepatocytes dependent on their position within a lobule. Proximal periportal and pericentral zones from liver lobules were isolated by LCM from formalin fixed mouse liver sections derived from a time-course treatment study with the non-genotoxic carcinogen TCPOBOP. About 200 Western-Blot equivalents were performed with the resulting samples. The analysis of the isolated zones provided new insights into cellular signaling in liver zonation and changes occurring during the treatment time course. An important role for serine and threonine phosphorylations and phosphatases was found for regulation of zone specific metabolism. Inhibition of the phosphatase PP2A in the periportal zone was found to direct insulin dependent signaling. The TCPOBOP treatment time-course analysis showed that mainly the pericentral zone is exposed to oxidative stress resulting in activation of a stress-survival response while other TCPOBOP induced effects were found to affect both zones. TCPOBOP was found to induce a pronounced disturbance in cellular signaling within the first 24 hours after treatment.
Western-Blot
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