NGFN-PLUS
Development of the scientific platform „Interaktom“ for systematic protein interaction studies
| Coordinator: | Prof. Dr. Erich Wanker | |
| Institution: | Max-Delbrück-Centrum für Molekulare Medizin (MDC) Berlin-Buch | |
| Homepage: | www.mdc-berlin.de |
To increase the understanding of the mechanisms of neurodegenerative disease processes (NDs) such as Alzheimer's, Parkinson's or Huntington's disease at the molecular level and to find new therapeutic approaches, it is crucial to identify new interaction partners of neurodegenerative disease proteins (NDPs) and to link them to known cellular processes and signaling cascades. To enable systematic protein interaction studies in high throughput (HT), the “Technology Platform Interactome" was established as an extension of the IG "NeuroNet".
The platform provided the technological foundation methods to identify and validate protein-protein interactions (PPIs) in a high-throughput manner. New pipetting robots were purchased, the processes of our existing automated Y2H method have been revised and improved; standardized protocols were established. Efficient measurement procedures have been developed. As a result, we are now able for the first time to test hundreds of thousands of PPIs per week.
Our high-content screening microscope (Cellomics Array Scan VTI) has been expanded to optimize the automated HT compound and PPI screens and cell culture studies: The microscope was equipped with an emission filter wheel and appropriate filters. We are now also able to perform HT-FRET measurements. The robot ensures the automatic delivery and removal of the microtiter plates. Furthermore, we performed a comprehensive update of the measurement and server software for a significant increase in the stability of the measurements.
In collaboration with the bioinformatics group of Dr. Andrade, different methods to investigate the influence of cellular changes on the formation and stability of protein complexes were established. Software-based tools were used for generation and analysis of PPI networks of cellular processes to predict functional units (modules) efficiently and with high precision. In addition, concepts and programs have been developed to combine PPI data with information of HT expression and localization studies carried out by collaborating research groups, especially from IG NeuroNet.
The platform provided the technological foundation methods to identify and validate protein-protein interactions (PPIs) in a high-throughput manner. New pipetting robots were purchased, the processes of our existing automated Y2H method have been revised and improved; standardized protocols were established. Efficient measurement procedures have been developed. As a result, we are now able for the first time to test hundreds of thousands of PPIs per week.
Our high-content screening microscope (Cellomics Array Scan VTI) has been expanded to optimize the automated HT compound and PPI screens and cell culture studies: The microscope was equipped with an emission filter wheel and appropriate filters. We are now also able to perform HT-FRET measurements. The robot ensures the automatic delivery and removal of the microtiter plates. Furthermore, we performed a comprehensive update of the measurement and server software for a significant increase in the stability of the measurements.
In collaboration with the bioinformatics group of Dr. Andrade, different methods to investigate the influence of cellular changes on the formation and stability of protein complexes were established. Software-based tools were used for generation and analysis of PPI networks of cellular processes to predict functional units (modules) efficiently and with high precision. In addition, concepts and programs have been developed to combine PPI data with information of HT expression and localization studies carried out by collaborating research groups, especially from IG NeuroNet.
Figure 1: K2 spotting robot: spotting of proteins on membranes
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