From Genome to Proteome: Looking at a Cell's Proteins

Kahn, Patricia

doi:10.1126/science.270.5235.369

Cited by 164 publications

(75 citation statements)

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“…Two-dimensional gel polyacrylamide electrophoresis (twodimensional PAGE) 1 is arguably one of the most powerful methodologies for the description of protein phenotypes (2,3), because it can separate and quantitate thousands of individual proteins from complex biological samples. The potential of twodimensional PAGE would be extended if good quality affinity reagents (e.g.…”

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confidence: 99%

Design and Use of a Phage Display Library

Pini¹,

Viti²,

Santucci³

et al. 1998

Journal of Biological Chemistry

397

108

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We report the construction and the use of a phage display human antibody library (>3 ؋ 10 8 clones) based on principles of protein design. A large repertoire of functional antibodies with similar properties was produced by appending short variable complementaritydetermining region 3 (CDR3) onto the two antibody germ line segments most frequently found in human antibodies. With this strategy we concentrated sequence diversity in regions of the antibody structure that are centrally located in the antigen binding site, while leaving residues in more peripheral positions available for further mutagenesis aimed at improving the affinity of the selected antibodies.In addition, the library was tested by selecting antibodies against six biologically relevant antigens. Using only 0.3 g of antigen eluted from a two-dimensional gel spot, we isolated binders specific for the ED-B domain of fibronectin, a marker of angiogenesis. These antibodies recognize the native antigen with affinities in the 10 7 -10 8 M ؊1 range, and perform well in immunosorbent assays, in two-dimensional Western blotting and in immunohistochemistry. The affinity of one anti-ED-B antibody was improved by 27-fold by combinatorially mutating six strategically selected residues in the heavy chain variable domain. A further 28-fold affinity improvement could be achieved by mutating residues 32 and 50 of the light chain. The resulting antibody, L19, bound to the ED-B domain of fibronectin with very high affinity (K d ‫؍‬ 54 pM), as determined by real-time interaction analysis with surface plasmon resonance detection, band shift analysis, and by competition experiments with electrochemiluminescent detection.

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mentioning

confidence: 99%

Design and Use of a Phage Display Library

Pini¹,

Viti²,

Santucci³

et al. 1998

Journal of Biological Chemistry

397

108

View full text Add to dashboard Cite

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“…2-D PAGE can be used to link certain gel patterns to specific diseases or modifications of specific marker proteins, and this technique can also be applied to answer molecular epidemiological questions in medical microbiology (1,4,7,10,11,15,16,19,20,25). The studies mentioned represent some examples of the wide variety of organisms and questions concerning bacterial protein expression that may be analyzed by the 2-D gel technique.…”

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confidence: 99%

Clustering of Clinical Strains of Helicobacter pylori Analyzed by Two-Dimensional Gel Electrophoresis

Enroth

Åkerlund

Sillén

et al. 2000

Clin Diagn Lab Immunol

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Strain variations of Helicobacter pylori have been tested by numerous methods and compared among different patient groups. The aim of this study was to investigate whether H. pylori expresses disease-specific proteins that can be detected by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). H. pylori strains isolated from duodenal ulcer, gastric cancer, and gastritis patients were analyzed. Extensive variation in spot patterns was observed between the strains, but a dendrogram analysis revealed that some strains within each disease group clustered together. Eight proteins were sequenced and found in the H. pylori genome sequence. 2-D PAGE is a useful method for studies of protein expression and for highlighting the extensive strain variation that H. pylori exhibits.

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“…It reveals different protein expression patterns under different dynamic situations, e.g. in normal and disease conditions they reflect the differences in molecular pathways [2,3]. Many factors may influence the expression pattern of the proteins, among them stress, alterations in temperature, different growth conditions, and the effects of various treatments.…”

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confidence: 99%

Two‐dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry

2006

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The proteome analysis by 2-DE is one of the most potent methods of analyzing the complete proteome of cells, cell lines, organs and tissues in proteomics studies. It allows a fast overview of changes in cell processes by analysis of the entire protein extracts in any biological and medical research projects. New instrumentation and advanced technologies provide proteomics studies in a wide variety of biological and biomedical questions. Proteomics work is being applied to study antibiotics-resistant strains and human tissues of various brain, lung, and heart diseases. It cumulated in the identification of antigens for the design of new vaccines. These advances in proteomics have been possible through the development of advanced high-resolution 2-DE systems allowing resolution of up to 10 000 protein spots of entire cell lysates in combination with protein identification by new highly sensitive mass spectrometric techniques. The present technological achievements are suited for a high throughput screening of different cell situations. Proteomics may be used to investigate the health effects of radiation and electromagnetic field to clarify possible dangerous alterations in human beings.

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From Genome to Proteome: Looking at a Cell's Proteins

Cited by 164 publications

References 0 publications

Design and Use of a Phage Display Library

Design and Use of a Phage Display Library

Clustering of Clinical Strains of Helicobacter pylori Analyzed by Two-Dimensional Gel Electrophoresis

Two‐dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry

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