Book of Abstracts - Ruhr-Universität Bochum

P-26
ISBOMC `10 5.7 – 9.7. 2010 Ruhr-Universität Bochum
Influence of Cisplatin and Other (Bio-)organometalic Compounds on Changes in
Cellular Signaling Using Novel Phosphoprotein Microarray Elisa Assays
Pavlo Holenya, a Igor Kitanovic, a and Stefan Wölfl *a
Institute of Pharmacy and Molecular Biotechnology, University Heidelberg, Germany
Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany
E-mail: wolfl@uni-hd.de
Understanding aspects of the regulatory network of signaling pathways and their role in the
development of the diseases is one of the crucial starting points in drug research. Signal transduction
in cells is regulated, among other things, by phosphorylation and dephosphorylation of numerous
signaling proteins. In many cases, phosphorylation reflects the activation state of those proteins within
pathways that control various biological responses. In order to understand how drugs influence cells
by modulating different pathways, it is highly desirable to simultaneously measure the
phosphorylation states of diverse signaling proteins and temporarily monitore their levels in cells.
For this we used a novel technology based on protein microarray platforms ArrayTube TM and
ArrayStrip TM developed by CLONDIAG Chip Technologies, Jena. Both platforms represent less
expensive and easy to handle systems for the development of protein arrays. The heart of the device is
a chemically modified glass surface assembled to form the bottom of a 1.5 ml plastic polystyrol
microtube or a standard well of a 96-well plate. Capture antibodies are deposited onto agarose-film
coated glass surface by contact spotting. Handling of the protein chip is easy and rapid and involves
the simple steps of an ELISA in a sandwich format. Specific interactions of antibody and antigen are
simply revealed by colorimetric detection. Acquisition and analysis of processed chip images are
performed by optical transmission microscopy in combination with image analysis software.
Using the ArrayTube TM and ArrayStrip TM platforms and commercially available capture and detection
antibodies, we established a protocol to quantitatively analyze changes in protein phosphorylation
upon treatment with diverse organometallic compounds. We demonstrate high sensitivity and
specificity of the microarrays and show quantitative analysis of several phosphorylated proteins,
among them phospho GSK-3β, phospho RSK1, phospho Akt1, phospho p38α, phospho Erk1, phospho
Erk2, phospho CREB, phospho TOR, phospho Src, phospho JNK, phospho p53, phospho STAT3,
phospho ATM.
Our results show that these newly developed arrays enable reliable evaluation of multiple target
protein phosphorylation in a single sample. The method requires minimal sample volumes (~10 μl)
and minimal amount of total protein (~1 μg) to obtain quantitative data, and it enables rapid evaluation
of multiple analytes.
We thank Clondiag GmbH, Jena, Germany for providing the microarray technology and the
colleagues from the DFG-Forschergruppe FOR630 for bioorganometallic substances.
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