Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form

Schägger, Hermann; Jagow, Gebhard von

doi:10.1016/0003-2697(91)90094-a

Cited by 2,093 publications

(1,628 citation statements)

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“…To conduct Blue native polyacrylamide gel electrophoresis (BN-PAGE) analyses (Schagger and von Jagow, 1991), protein complexes were first solubilised in the presence of either N-dodecyl-β-Dmaltoside (0.25-1.5%), digitonin (0.5-3.5%) (w/v), 750 mM epsilon-amino-n-caproic acid, 0.5 mM EDTA and 50 mM Bis-Tris pH 7.0, and centrifuged for 15 min at 15,000 g at 4°C to remove insoluble matter. 0.5% (w/v) sodium taurodeoxycholate was then added to the supernatant prior to separation by electrophoresis on a 4-10% or 4-12% (w/v) polyacrylamide gradient BN gel.…”

Section: Protein Electrophoresismentioning

confidence: 99%

The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae

et al. 2014

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The mitochondrion is an essential organelle for the production of cellular ATP in most eukaryotic cells. It is extensively studied, including in parasitic organisms such as trypanosomes, as a potential therapeutic target. Recently, numerous additional subunits of the respiratory-chain complexes have been described in Trypanosoma brucei and Trypanosoma cruzi. Since these subunits had apparently no counterparts in other organisms, they were interpreted as potentially associated with the parasitic trypanosome lifestyle. Here we used two complementary approaches to characterise the subunit composition of respiratory complexes in Euglena gracilis, a non-parasitic secondary green alga related to trypanosomes. First, we developed a phylogenetic pipeline aimed at mining sequence databases for identifying homologs to known respiratory-complex subunits with high confidence. Second, we used MS/MS proteomics after two-dimensional separation of the respiratory complexes by Blue Native-and SDS-PAGE to both confirm in silico predictions and to identify further additional subunits. Altogether, we identified 41 subunits that are restricted to E. gracilis, T. brucei and T. cruzi, along with 48 classical subunits described in other eukaryotes (i.e. plants, mammals and fungi). This moreover demonstrates that at least half of the subunits recently reported in T. brucei and T. cruzi are actually not specific to Trypanosomatidae, but extend at least to other Euglenozoa, and that their origin and function are thus not specifically associated with the parasitic lifestyle. Furthermore, preliminary biochemical analyses suggest that some of these additional subunits underlie the peculiarities of the respiratory chain observed in Euglenozoa.Liège, february 06 th 2014Dear Editor, Please find the fully revised version of our manuscript "The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae."We are grateful to you for having encouraged us to resubmit a modified manuscript. We have addressed all the comments raised by the reviewers on our first submission. A point to point answer has been submitted along with the manuscript. Main changes are : (i) additional explanations about the phylogenic/bioinformatic strategy including some phylogenetic trees (supplemental figures 1 and 2); (ii) all data that were previously not shown (mass spectrometry data, in vivo respiratory assays) are now included in the manuscript as supplemental figures/tables.We hope that we have now significantly improved our manuscript so you'll find it suitable for publication in the special issue of Mitochondrion dedicated to "Plant Mitochondria Biology". R: Primary MS data are now given in Supplemental Table 3 for each polypeptide for which the score was > 60. (BLAST alignments, Evalues, etc.) are actually presented in the paper to support the inference that a particular E. gracilis protein is a true homolog of a given kinetoplastid protein. The most important conclusion of t...

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Section: Protein Electrophoresismentioning

confidence: 99%

The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae

et al. 2014

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“…The initial supernatant from the solubilization of the membranes and the purified enzyme were analysed by Blue Native PAGE as described previously [26]. This gel was sliced into individual lanes for histochemical staining.…”

Section: In Gel Activity Assay Of F 1 F 0 Atp Synthasementioning

confidence: 99%

Biochemical and electron microscopic characterization of the F₁F₀ ATP Synthase from the hyperthermophilic eubacterium Aquifex aeolicus

et al. 2006

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The F 1 F 0 ATP synthase has been purified from the hyperthermophilic eubacterium Aquifex aeolicus and characterized. Its subunits have been identified by MALDI-mass spectrometry through peptide mass fingerprinting and MS/MS. It contains the canonical subunits a, b, c, d and e of F 1 and subunits a and c of F 0 . Two versions of the b subunit were found, which show a low sequence homology to each other. Most likely they form a heterodimer. An electron microscopic single particle analysis revealed clear structural details, including two stalks connecting F 1 and F 0 . In several orientations the central stalk appears to be tilted and/or kinked. It is unclear whether there is a direct connection between the peripheral stalk and the d subunit.

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“…Blue native PAGE was performed essentially as described (12,31,32). Briefly, COS-7 mitochondrial pellets (50 -100 g of protein) were lysed in 50 l of ice-cold digitonin buffer (1% digitonin, 20 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 50 mM NaCl, 10% glycerol, 1 mM phenylmethyl sulfonyl fluoride).…”

Section: Preparation Of Mitochondria From Cos-7 Cells Cos-7 Cellsmentioning

confidence: 99%

Functional Analysis of Human Metaxin in Mitochondrial Protein Import in Cultured Cells and Its Relationship with the Tom Complex

Abdul

Terada

Yano

et al. 2000

Biochemical and Biophysical Research Communications

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Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form

Cited by 2,093 publications

References 17 publications

The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae

The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae

Biochemical and electron microscopic characterization of the F₁F₀ ATP Synthase from the hyperthermophilic eubacterium Aquifex aeolicus

Functional Analysis of Human Metaxin in Mitochondrial Protein Import in Cultured Cells and Its Relationship with the Tom Complex

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Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form

Cited by 2,093 publications

References 17 publications

The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae

The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae

Biochemical and electron microscopic characterization of the F1F0 ATP Synthase from the hyperthermophilic eubacterium Aquifex aeolicus

Functional Analysis of Human Metaxin in Mitochondrial Protein Import in Cultured Cells and Its Relationship with the Tom Complex

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Biochemical and electron microscopic characterization of the F₁F₀ ATP Synthase from the hyperthermophilic eubacterium Aquifex aeolicus