Alkaline proteins ofBacillus subtilis: First steps towards a two-dimensional alkaline master gel

Ohlmeier, Steffen; Scheffold, Christian; Hecker, Michael

doi:10.1002/1522-2683(200011)21:17<3701::aid-elps3701>3.0.co;2-5

Cited by 39 publications

(9 citation statements)

References 39 publications

(42 reference statements)

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“…The PMF of spot 2 and spot 3 showed the best fit to heat shock protein 70 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively, which are known cytosolic soluble proteins ( Table 1), suggesting that the supernatant fraction contained soluble proteins. Many membrane-associated proteins are known to possess a high pI [12,13], which is consistent with the data shown in Fig. 3.…”

Section: Subcellular Fractionation Using Protoplastssupporting

confidence: 90%

Development of a sample preparation method for fungal proteomics

Shimizu¹,

Wariishi²

2005

FEMS Microbiology Letters

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Since filamentous fungi including basidiomycetous fungi possess an exceptionally robust cell wall as in microorganisms, effective extraction of intracellular proteins is a key step for fungal proteomic studies. To overcome the experimental obstacle caused by cell walls, we utilized fungal protoplasts, prepared from the brown-rot basidiomycete, Tyromyces palustris. The amount and quality of proteins extracted from the protoplast cells were much higher than that from the mycelial cells. Quantitative comparisons of proteome maps prepared from mycelial and protoplast cells indicated protein spots with a wider range of molecular weights and pIs in the protoplast sample. Furthermore, no streaking or tailing was observed in the protoplasts, suggesting that effective extraction of intracellular proteins from protoplasts might help suppress degradation of proteins during this process. In addition to the efficiency of protein extraction, simple and efficient subcellular fractionation was also achieved using protoplast cells.

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Section: Subcellular Fractionation Using Protoplastssupporting

confidence: 90%

Development of a sample preparation method for fungal proteomics

Shimizu¹,

Wariishi²

2005

FEMS Microbiology Letters

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“…The limited capacity of 2-DE for the analysis of membrane and membrane-associated proteins has primarily been attributed to obstacles related to their extraction, solubilization, and low abundance [38,67]. In addition, many integral membrane proteins have highly alkaline pI values (.10) preventing their detection by conventional 2-DE [38,68]. This also holds true for many integral plasma membrane proteins of M. tuberculosis H37Rv (Figure S3, Supporting Information).…”

Section: Discussionmentioning

confidence: 99%

An improved strategy for selective and efficient enrichment of integral plasma membrane proteins of mycobacteria

et al. 2007

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Mycobacterial plasma membrane proteins play essential roles in many cellular processes, yet their comprehensive proteomic profiling remains challenging. This is mainly due to obstacles related to their extraction and solubilization. To tackle this problem, we have developed a novel procedure to selectively enrich mycobacterial plasma membrane proteins based on alkaline sodium carbonate washing of crude membranes followed by Triton X-114 phase partitioning. The present study assesses the efficiency of this method by proteome analysis of plasma membrane proteins from Mycobacterium bovis BCG. Extracted proteins were separated in parallel by 1-D SDS-PAGE and 2-DE and then analyzed by LC-MS/MS and MALDI-MS/MS. Our study revealed 125 proteins, of which 54 contained 1-14 predicted transmembrane domains (TMD) including nine novel proteins. The 1-D SDS-PAGE-based proteome analysis identified 81 proteins, of which 49 (60.5%) harbored TMD. This approach also revealed many hydrophobic membrane-associated/periplasmic proteins lacking TMD, but only few soluble proteins. The identified proteins were characterized with regard to biological functions and physicochemical properties providing further evidence for the high efficiency of the prefractionation method described herein.

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“…These are usually not separated because in the first dimension the pH gradient used is usually 4-7. It is possible to separate the alkaline proteins (with lower resolution) on wide pH-gradient gels (Ohlmeier et al 2000). The third group of proteins that is not easily studied on 2D gel is the rare proteins.…”

Section: Proteome Analysismentioning

confidence: 99%

Molecular tools in rhizosphere microbiology—from single-cell to whole-community analysis

et al. 2009

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It is the aim of this chapter to present an overview of new, molecular tools that have been developed over recent years to study individual, single cells and composite, complex communities of microorganisms in the rhizosphere. We have carefully focused on culture-independent assays and selected methodologies that have already been or will soon be applicable for rhizosphere microbiology. Emphasis is placed on rhizosphere bacteria and the review first describes a number of the new methodologies developed for detection and localization of specific bacterial populations using modern electron and fluorescence microscopy combined with specific tagging techniques. First half of the chapter further comprises a thorough treatise of the recent development of reporter gene technology, i.e. using specific reporter bacteria to detect microscale distributions of rhizosphere compounds such as nutrients, metals and organic exudates or contaminants. Second half of the chapter devoted to microbial community analysis contains a thorough treatise of nucleotide-and PCRbased technologies to study composition and diversity of indigenous bacteria in the natural rhizosphere. Also included are the most recent developments of functional gene and gene expression analyses in the rhizosphere based on specific mRNA transcript or transcriptome analysis, proteome analysis and construction of metagenomic libraries.

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Alkaline proteins ofBacillus subtilis: First steps towards a two-dimensional alkaline master gel

Cited by 39 publications

References 39 publications

Development of a sample preparation method for fungal proteomics

Development of a sample preparation method for fungal proteomics

An improved strategy for selective and efficient enrichment of integral plasma membrane proteins of mycobacteria

Molecular tools in rhizosphere microbiology—from single-cell to whole-community analysis

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