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Simultaneous isolation of pure and intact chloroplasts and mitochondria from moss as the basis for sub-cellular proteomics.

Lang EG, Mueller SJ, Hoernstein SN, Porankiewicz-Asplund J, Vervliet-Scheebaum M, Reski R - Plant Cell Rep. (2010)

Bottom Line: In the present study we describe a rapid and efficient protocol for the simultaneous isolation of chloroplasts and mitochondria from moss protonema.Routinely, 60-100 μg mitochondrial and 3-5 mg chloroplast proteins, respectively, were obtained from 20 g fresh weight of green moss tissue.Using 14 plant compartment marker antibodies derived from seed plant and algal protein sequences, respectively, the evolutionary conservation of the compartment marker proteins in the moss was demonstrated and purity and intactness of the extracted organelles confirmed.

View Article: PubMed Central - PubMed

Affiliation: Plant Biotechnology, Faculty of Biology, University of Freiburg, Schaenzlestr 1, 79104 Freiburg, Germany.

ABSTRACT
The moss Physcomitrella patens is increasingly being used as a model for plant systems biology studies. While genomic and transcriptomic resources are in place, tools and experimental conditions for proteomic studies need to be developed. In the present study we describe a rapid and efficient protocol for the simultaneous isolation of chloroplasts and mitochondria from moss protonema. Routinely, 60-100 μg mitochondrial and 3-5 mg chloroplast proteins, respectively, were obtained from 20 g fresh weight of green moss tissue. Using 14 plant compartment marker antibodies derived from seed plant and algal protein sequences, respectively, the evolutionary conservation of the compartment marker proteins in the moss was demonstrated and purity and intactness of the extracted organelles confirmed. This isolation protocol and these validated compartment markers may serve as basis for sub-cellular proteomics in P. patens and other mosses.

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Immunoblots assessing the organellar purity of urea-extracted proteins. T total protein, C chloroplast protein, M1 mitochondrial fraction 1 protein, M2 mitochondrial fraction 2 protein, loading amounts: 10 μg/lane (a, e), 2.5 μg/lane (b–d, f, g); for dilutions of primary antibodies, see Table 1
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Fig3: Immunoblots assessing the organellar purity of urea-extracted proteins. T total protein, C chloroplast protein, M1 mitochondrial fraction 1 protein, M2 mitochondrial fraction 2 protein, loading amounts: 10 μg/lane (a, e), 2.5 μg/lane (b–d, f, g); for dilutions of primary antibodies, see Table 1

Mentions: Impurities in the mitochondrial fraction were assessed using antibodies against plastidic contaminants, namely, the membrane localised light-harvesting complex II type II chlorophyll a/b-binding protein (Lhcb2) and the plastidic stroma localised glutamine synthetase (GLN1/GLN2), and antibodies against Golgi apparatus and tonoplast membrane contaminants, namely, the ADP-ribosylation factor 1 (Arf1) and the Epsilon subunit of the tonoplast H+ ATPase (V-type ATPase) localised in Golgi and tonoplast, respectively (Fig. 3).Fig. 3


Simultaneous isolation of pure and intact chloroplasts and mitochondria from moss as the basis for sub-cellular proteomics.

Lang EG, Mueller SJ, Hoernstein SN, Porankiewicz-Asplund J, Vervliet-Scheebaum M, Reski R - Plant Cell Rep. (2010)

Immunoblots assessing the organellar purity of urea-extracted proteins. T total protein, C chloroplast protein, M1 mitochondrial fraction 1 protein, M2 mitochondrial fraction 2 protein, loading amounts: 10 μg/lane (a, e), 2.5 μg/lane (b–d, f, g); for dilutions of primary antibodies, see Table 1
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3020298&req=5

Fig3: Immunoblots assessing the organellar purity of urea-extracted proteins. T total protein, C chloroplast protein, M1 mitochondrial fraction 1 protein, M2 mitochondrial fraction 2 protein, loading amounts: 10 μg/lane (a, e), 2.5 μg/lane (b–d, f, g); for dilutions of primary antibodies, see Table 1
Mentions: Impurities in the mitochondrial fraction were assessed using antibodies against plastidic contaminants, namely, the membrane localised light-harvesting complex II type II chlorophyll a/b-binding protein (Lhcb2) and the plastidic stroma localised glutamine synthetase (GLN1/GLN2), and antibodies against Golgi apparatus and tonoplast membrane contaminants, namely, the ADP-ribosylation factor 1 (Arf1) and the Epsilon subunit of the tonoplast H+ ATPase (V-type ATPase) localised in Golgi and tonoplast, respectively (Fig. 3).Fig. 3

Bottom Line: In the present study we describe a rapid and efficient protocol for the simultaneous isolation of chloroplasts and mitochondria from moss protonema.Routinely, 60-100 μg mitochondrial and 3-5 mg chloroplast proteins, respectively, were obtained from 20 g fresh weight of green moss tissue.Using 14 plant compartment marker antibodies derived from seed plant and algal protein sequences, respectively, the evolutionary conservation of the compartment marker proteins in the moss was demonstrated and purity and intactness of the extracted organelles confirmed.

View Article: PubMed Central - PubMed

Affiliation: Plant Biotechnology, Faculty of Biology, University of Freiburg, Schaenzlestr 1, 79104 Freiburg, Germany.

ABSTRACT
The moss Physcomitrella patens is increasingly being used as a model for plant systems biology studies. While genomic and transcriptomic resources are in place, tools and experimental conditions for proteomic studies need to be developed. In the present study we describe a rapid and efficient protocol for the simultaneous isolation of chloroplasts and mitochondria from moss protonema. Routinely, 60-100 μg mitochondrial and 3-5 mg chloroplast proteins, respectively, were obtained from 20 g fresh weight of green moss tissue. Using 14 plant compartment marker antibodies derived from seed plant and algal protein sequences, respectively, the evolutionary conservation of the compartment marker proteins in the moss was demonstrated and purity and intactness of the extracted organelles confirmed. This isolation protocol and these validated compartment markers may serve as basis for sub-cellular proteomics in P. patens and other mosses.

Show MeSH