Kurt Dejgaard

Human 2-D PAGE databases for proteome analysis in health and disease: http ://biobase.dk/cgi-bin/celis

Human 2‐D PAGE Databases established at the Danish Centre for Human Genome Research are now available on the World Wide Web (http://biobase.dk/cgi‐bin/celis). The databanks, which offer a comprehensive approach to the analysis of the human proteome both in health and disease, contain data on known and unknown proteins recorded in various IEF and NEPHGE 2‐D PAGE reference maps (non‐cultured keratinocytes, non‐cultured transitional cell carcinomas, MRC‐5 fibroblasts and urine). One can display names and information on specific protein spots by clicking on the image of the gel representing the 2‐D gel map in which one is interested. In addition, the database can be searched by protein name, keywords or organelle or cellular component. The entry files contain links to other databases such as Medline, Swiss‐Prot, PIR, PDB, CySPID, OMIM, Methabolic pathways, etc. The on‐line information is updated regularly.

Human cellular protein patterns and their link to genome DNA sequence data: Usefulness of two-dimensional gel electrophoresis and microsequencing

Analysis of cellular protein patterns by computer‐aided 2‐dimensional gel electrophoresis together with recent advances in protein sequence analysis have made possible the establishment of comprehensive 2‐dimensional gel protein databases that may link protein and DNA information and that offer a global approach to the study of the cell. Using the integrated approach offered by 2‐dimensional gel protein databases it is now possible to reveal phenotype specific protein (or proteins), to microsequence them, to search for homology with previously identified proteins, to clone the cDNAs, to assign partial protein sequence to genes for which the full DNA sequence and the chromosome location is known, and to study the regulatory properties and function of groups of proteins that are coordinately expressed in a given biological process. Human 2‐dimensional gel protein databases are becoming increasingly important in view of the concerted effort to map and sequence the entire genome.—Celis, J. E.; Rasmussen, H. H.; Leffers, H.; Madsen, P.; Honoré, B.; Gesser, B.; Dejgaard, K.; Vandekerckhove, J. Human cellular protein patterns and their link to genome DNA sequence data: usefulness of two‐dimensional gel electrophoresis and microsequencing. FASEB J. 5: 2200–2208; 1991.

Two-Dimensional Northwestern Blotting

Publisher Summary This chapter focuses on two-dimensional (2D) Northwestern blotting. 2D Northwestern blotting complements gel-shift experiments in the identification of specific protein–nucleic acid interactions. As the blocking buffer is protein free, the blot is stained after exposure with amido black or Coomassie brilliant blue to determine the molecular weight and isoelectric point of the binding proteins. However, there are two main reasons why Northwestern blotting cannot substitute for gel-shift experiments or native affinity chromatography for the analysis of protein–nucleotide interactions. The protein may not have the DNA/RNA binding site reconstituted after sodium dodecyl sulfate electrophoresis and blotting and the protein may only bind to DNA/RNA when interacting with other proteins to form a hetero oligomer. Although Northwestern blotting works for many proteins, it provides no information on other proteins with which the DNA- or RNA-binding proteins may be associated under native conditions.

Human Cellular Protein Patterns and Their Link to Genome DNA Mapping and Sequencing Data: Towards an Integrated Approach to the Study of Gene Expression

Analysis of cellular protein patterns by computer-aided two-dimensional gel electrophoresis together with recent advances in protein sequence analysis and expression systems have made possible the establishment of comprehensive two-dimensional gel protein databases that may link protein and DNA mapping and sequence information and that offer an integrated approach to the study of gene expression. With the integrated approach offered by two-dimensional gel protein databases it is now possible to reveal phenotype-specific protein(s), to microsequence them, to search for homology with previous identified proteins, to clone the cDNAs, to assign partial protein sequences to genes for which the full DNA sequence and the chromosome location are known, and to study the regulatory properties and function of groups of proteins that are coordinately expressed in a given biological process. Comprehensive two-dimensional gel protein databases will provide an integrated picture of the expression levels and properties of the thousands of protein components of organelles, pathways, and cytoskeletal systems, both under physiological and abnormal conditions, and are expected to lead to the identification of new regulatory networks. So far, about 20% (600 out of 2,980) of the total number of proteins recorded in the human keratinocyte protein database have been identified and we are actively gathering qualitative and quantitative biological data on all resolved proteins. Given the current improvements on microsequencing as well as the availability of specific antibodies, it seems feasible to expect that most known keratinocyte proteins will be identified in the very near future. This feast will reveal a wealth of new proteins that will become amenable to experimentation both at the biochemical and molecular biology level.