Blue-native PAGE in plants: a tool in analysis of protein-protein interactions

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract

Intact protein complexes can be separated by apparent molecular mass using a standard polyacrylamide gel electrophoresis system combining mild detergents and the dye Coomassie Blue. Referring to the blue coloured gel and the gentle method of solubilization yielding native and enzymatically active protein complexes, this technique has been named Blue-Native Polyacrylamide Gel-Electrophoresis (BN-PAGE). BN-PAGE has become the method of choice for the investigation of the respiratory protein complexes of the electron transfer chains of a range of organisms, including bacteria, yeasts, animals and plants. It allows the separation in two dimensions of extremely hydrophobic protein sets for analysis and also provides information on their native interactions. In this review we discuss the capabilities of BN-PAGE in proteomics and the wider investigation of protein:protein interactions with a focus on its use and potential in plant science.

Related collections

Most cited references 120

Record: found
Abstract: found
Article: not found

A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae.

Peter Uetz, Loic Giot, Gerard Cagney … (2000)

Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.

0 comments Cited 899 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

A comprehensive two-hybrid analysis to explore the yeast protein interactome.

T Ito, T Chiba, R Ozawa … (2001)

Protein-protein interactions play crucial roles in the execution of various biological functions. Accordingly, their comprehensive description would contribute considerably to the functional interpretation of fully sequenced genomes, which are flooded with novel genes of unpredictable functions. We previously developed a system to examine two-hybrid interactions in all possible combinations between the approximately 6,000 proteins of the budding yeast Saccharomyces cerevisiae. Here we have completed the comprehensive analysis using this system to identify 4,549 two-hybrid interactions among 3,278 proteins. Unexpectedly, these data do not largely overlap with those obtained by the other project [Uetz, P., et al. (2000) Nature (London) 403, 623-627] and hence have substantially expanded our knowledge on the protein interaction space or interactome of the yeast. Cumulative connection of these binary interactions generates a single huge network linking the vast majority of the proteins. Bioinformatics-aided selection of biologically relevant interactions highlights various intriguing subnetworks. They include, for instance, the one that had successfully foreseen the involvement of a novel protein in spindle pole body function as well as the one that may uncover a hitherto unidentified multiprotein complex potentially participating in the process of vesicular transport. Our data would thus significantly expand and improve the protein interaction map for the exploration of genome functions that eventually leads to thorough understanding of the cell as a molecular system.

0 comments Cited 806 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry.

Yuen Ho, Albrecht Gruhler, Adrian Heilbut … (2002)

The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks.

0 comments Cited 728 times – based on 0 reviews      Review now

Bookmark

All references

Author and article information

Journal

Journal ID (nlm-ta): Plant Methods

Title: Plant Methods

Publisher: BioMed Central (London )

ISSN (Electronic): 1746-4811

Publication date Collection: 2005

Publication date (Electronic): 16 November 2005

Volume: 1

Page: 11

Affiliations

[1 ]ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, 35 Stirling Hwy, Crawley 6009, Perth, Australia

[2 ]Abteilung Angewandte Genetik, Universität Hannover, Herrenhäuser Str. 2, 30419 Hannover, Germany

Article

Publisher ID: 1746-4811-1-11

DOI: 10.1186/1746-4811-1-11

PMC ID: 1308860

PubMed ID: 16287510

SO-VID: a74a7cd0-c6dc-4ed1-8bee-54ade57a2732

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Blue-native PAGE in plants: a tool in analysis of protein-protein interactions

Read this article at

Abstract

Related collections

Plant MYBs

Most cited references 120

A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae.

A comprehensive two-hybrid analysis to explore the yeast protein interactome.

Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry.

Author and article information

Journal

Affiliations

Article

History

Categories

Comments

Comment on this article

Similar content 374

Cited by 31

Most referenced authors 2,038