Biomedical subjects

Gerald Walter

Publications and source records attributed to Gerald Walter.

4 recordsLinked to original sources

PubMed2003

Rapid identification of allergen-encoding cDNA clones by phage display and high-density arrays.

We describe a high-throughput, quantitative technology for fast identification of all different clones present in selectively enriched phage surface-displayed cDNA libraries. The strategy is based on a combination of phage display and high-density arrays. To demonstrate the utility of the method cDNAs of Aspergillus fumigatus cloned into phagemid pJuFo were expressed on the tip of filamentous M13 phage and affinity-selected on solid phase-immobilized serum IgE from allergic patients. Enriched phagemid libraries were amplified in bacteria, plated and arrayed into 384-well microtitre plates by robotic colony picking. cDNA inserts were amplified by high-throughput PCR and gridded onto high-density filter membranes. Filters were iteratively probed with randomly-sequenced inserts until all clones were identified. Eighty-one different sequences encoding IgE-binding proteins likely to cover a large part of the allergen repertoire of the mould were found. This approach represents a widely applicable method for rapid high-throughput identification of all individual cDNAs present in selectively enriched libraries.

Allergens↗

PubMed2002

Protein array technology: the tool to bridge genomics and proteomics.

The generation of protein chips requires much more efforts than DNA microchips. While DNA is DNA and a variety of different DNA molecules behave stable in a hybridisation experiment, proteins are much more difficult to produce and to handle. Outside of a narrow range of environmental conditions, proteins will denature, lose their three-dimensional structure and a lot of their specificity and activity. The chapter describes the pitfalls and challenges in Protein Microarray technology to produce native and functional proteins and store them in a native and special environment for every single spot on an array, making applications like antibody profiling and serum screening possible not only on denatured arrays but also on native protein arrays.

Databases, Protein↗

PubMed2002

High-throughput protein arrays: prospects for molecular diagnostics.

High-throughput protein arrays allow the miniaturized and parallel analysis of large numbers of diagnostic markers in complex samples. Using automated colony picking and gridding, cDNA or antibody libraries can be expressed and screened as clone arrays. Protein microarrays are constructed from recombinantly expressed, purified, and yet functional proteins, entailing a range of optimized expression systems. Antibody microarrays are becoming a robust format for expression profiling of whole genomes. Alternative systems, such as aptamer, PROfusion, nano- and microfluidic arrays are all at proof-of-concept stage. Differential protein profiles have been used as molecular diagnostics for cancer and autoimmune diseases and might ultimately be applied to screening of high-risk and general populations.

Animals↗

PubMed2002

Large-scale plant proteomics.

Large-scale and high throughput approaches increasingly play an essential role in the study of biological systems, which are per se highly complex. Therefore, they need to be examined by these extensive methods to receive information about the large genomic and proteomic networks. In plant biology, this purpose has a strong support through the accessability of the complete genome sequence of the model plant Arabidopsis thaliana. This brief review intends to focus on the basics and the state-of-the-art of these high-throughput technologies and their application to plant proteomics. It describes protein microarrays, the use of antibodies, 2-DE and MS methods and the yeast two hybrid system, which are emerging as the major technologies for plant proteomics.

Arabidopsis↗