Ziegler
Invention
The NAD group at the Department for Molecular Biology of the University of Bergen developed new technologies which significantly improve the sensitivity and accuracy of protein detection as well as a novel metabolite sensor were developed. The basic principle of these methods includes the enzymatic generation of large homo-polymers (poly-ADP-ribose) made from a small biomolecule (NAD+). Antibodies specifically recognizing these polymers are used for highly efficient immunochemical detection. Belowyou can read about three of the technologies developed by the NAD group.
PARAPLAY: High Resolution Protein Localization
One of the inventions is the PARAPLAY technology (Poly-ADP-Ribose-Assisted Protein Localization AssaY) which provides a unique tool to establish suborganellar protein localization and yet requires only the same experimental efforts as for GFP fusion proteins. Fusion proteins (for example, with GFP) are widely used to determine the subcellular distribution of proteins. However, they do not resolve whether a protein is associated with an organelle or resides in the lumen, a critical distinction to understand protein function.
The fusion of the analyte protein to a PARP (poly-ADP-ribose polymerase) will lead to polymer formation which can be detected as shown above. However, polymers can only be accumulated within organelles where the substrate (NAD+) is available and polymer degradation (by other cellular enzymes) is very slow. Besides luminal localization of proteins in various organelles (e.g., ER, Golgi, peroxisomes), PARAPLAY allows to distinguish between matrix and intermembrane proteins in mitochondria (Dölle et al. (2010), Cell. Mol. Life Sci. 67, 433-443).
Principle of polymer-enhanced immunodetection of proteins
One of the inventions is a novel strategy to amplify the signal in immunochemical protein detection methods. The new technology is based on the enzymatic generation of large polymers which provide a multitude of detectable epitopes.
In most conventional immunochemical applications (Western Blot, immunocytochemistry, ELISA etc.) an antigen (brown) is recognized by a primary antibody, which is then detected by a dye- or enzyme-coupled secondary antibody (left). The new method includes attachment of a polymerase to the primary antibody whose activity will generate a multitude of binding sites for a secondary, polymer-specific antibody.
In situ detection of NAD+
NAD+ is essential for energy metabolism, but also controls fundamental processes such as life span regulation, DNA repair and epigenetic modifications. Its subcellular distribution and local concentrations are crucial for these processes and any changes may have severe consequences. However, being a small molecule, NAD+ cannot be easily detected in cells. This novel approach overcomes this problem: When PARP activity is directed to the desired subcellular location, local NAD+ pools are converted to immunodetectable polymers. Changes in local NAD+ pools will therefore be reflected in differences of polymer abundance.
Interested in Ziegler’s technologies?
Contact buisness development manager Torsten Porwol.
