Eydfinnur Olsen

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 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.

One-Dimensional Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

Publisher Summary This chapter discusses one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis method. This method is one of the most used techniques for resolving and determining the apparent molecular mass of protein subunits. The SDS solubilizes insoluble proteins, making possible the analysis of otherwise insoluble mixtures. The method uses sterile, disposable pipettes to dispense solutions. If part of the bands does not move equally down the gel it is most likely because of air bubbles trapped between the plates at the bottom of the gel. The pH of the gel buffer should be checked carefully. Sterile, disposable pipettes should be used for dispensing the solutions. Moreover, all the glassware should be properly washed.