How to analyze protein complexes by 2D blue native SDS-PAGE.

Ludwig Maximilian University, Munich, Germany.
Proteomics (Impact Factor: 3.97). 09/2007; 7 Suppl 1(S1):6-16. DOI: 10.1002/pmic.200700205
Source: PubMed

ABSTRACT Natural compartmentalization makes proteome analysis of the cell, cell organelles and organelle subfractions possible. Protein complexes are the basis for the next level of compartmentalization that can be addressed well with proteomic technology. Protein complexes organize and maintain the cellular and organelle functions on all levels of complexity in time and space. Cell development and division, transcription and translation, respiration and photosynthesis, transport and metabolism can be defined by the activity of protein complexes. Since a large part of the protein complexes of the cell body are inserted in lipid membrane phases, isolation, separation and protein subunit identification were difficult to address. Blue native polyacrylamide gel electrophoresis (BN-PAGE) provides us with the technology for high resolution separation of membrane protein complexes. Here, we show that high resolution separation of protein complexes by BN-PAGE requires the establishment of a detailed solubilisation strategy. We show that BN/SDS-PAGE provides the scientist with a high resolution array of protein subunits which allows analysis of the specific subunit stoichiometry of a protein complex as well as the assembly of protein complexes by standard protein detection methodology like DIGE, gelblot analysis and mass spectrometry. We envision BN-PAGE to precede classical 2D IEF/SDS-analysis for detailed characterization of membrane proteomes.

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    • "2-DE blue native (BN)/SDS-PAGE analysis of thylakoid protein complexes Aliquots (200 ␮g) of pelleted chloroplasts isolated from three independent preparations (each from a different batch of 10 plants) from control and plants subjected to salt stress conditions , were suspended in ACA 750 (750 mM aminocaproic acid, 50 mM Bis–Tris pH 7.0, and 0.5 mM EDTA) and dissolved in 9 mM digitonine and 4.5 mM dodecyl maltoside for the extraction of intact thylakoid protein complexes. Protein complexes were separated under native conditions using BN PAGE (Reisinger and Eichacker, 2007) in 6–12% acrylamide gradient gels (Ruby SE 600 chamber, GE Healthcare). Electrophoresis was carried out at 4 • C and run at 45 V for 1 h and 160–180 V overnight (15–16 h). "
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    ABSTRACT: The effect of salt stress was analyzed in chloroplasts of Amaranthus cruentus var. Amaranteca, a plant NAD-malic enzyme (NAD-ME) type. Morphology of chloroplasts from bundle sheath (BSC) and mesophyll (MC) was observed by transmission electron microscopy (TEM). BSC and MC from control plants showed similar morphology, however under stress, changes in BSC were observed. The presence of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) was confirmed by immunohistochemical staining in both types of chloroplasts. Proteomic profiles of thylakoid protein complexes from BSC and MC, and their changes induced by salt stress were analyzed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (2-D BN/SDS-PAGE). Differentially accumulated protein spots were analyzed by LC-MS/MS. Although A. cruentus photosynthetic tissue showed the Kranz anatomy, the thylakoid proteins showed some differences at photosystem structure level. Our results suggest that A. cruentus var. Amaranteca could be better classified as a C3-C4 photosynthetic plant.
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    • "Já na segunda dimensão, ocorre uma separação das subunidades do complexo. Em ambas as dimensões, a separação ocorre primariamente pelo peso molecular (Reisinger & Eichacker 2007). O BN-PAGE fornece-nos a tecnologia necessária para uma separação em alta resolução dos complexos protéicos de membrana , permitindo a análise estequiométrica de uma subunidade especíϐica de um complexo protéico, tão bem como de um conjunto de complexos protéicos. "
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    ABSTRACT: The voltage dependent anion channel (VDAC) is the most abundant protein of outer mitochondrial membrane. VDAC controls metabolite exchange through this membrane and the apoptosis machinery. Interactomics is the study of protein complexes, their interactions and the consequences of these interactions. In our case we studied the interactome of the hexokinase-VDAC-ANT (adenine nucleotide transporter) complex in mitochondria of neuronal cells from rat, bovine and chicken brain. We wished to understand if the differential expression of VDAC1 and VDAC2 verified in these cells was linked to differences in the interactions between proteins in these complexes. Our results showed that avian and bovine neurons had more protein complexes (5) containing VDAC than rat cells (1), which indicates a differential kinetics of assembly or disassembly. Moreover, mitochondrial neuronal chicken VDAC interacts with more proteins in comparison with bovine neuronal VDAC, which is indicative of a differential subunit composition. These results supported evidences of differential apoptotic and energetic mechanisms between these brains.
    Pesquisa Veterinária Brasileira 11/2011; 31(11):1031-1038. DOI:10.1590/S0100-736X2011001100016 · 0.44 Impact Factor
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    • "The use of native PAGE has been amply described (e.g. Schä gger and von Jagow, 1991; Schä gger et al., 1994; Niepmann and Zheng, 2006; Wittig et al., 2006; Reisinger and Eichacker, 2007; Wittig and Schä gger, 2008). The composition of the different buffer systems is given in Table 1. "
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    ABSTRACT: Native polyacrylamide gel electrophoresis (PAGE) is an important technique for the analysis of membrane protein complexes. A major breakthrough was the development of blue native (BN-) and high resolution clear native (hrCN-) PAGE techniques. Although these techniques are very powerful, they could not be applied to all systems with the same resolution. We have developed an alternative protocol for the analysis of membrane protein complexes of plant chloroplasts and cyanobacteria, which we termed histidine- and deoxycholate-based native (HDN-) PAGE. We compared the capacity of HDN-, BN- and hrCN-PAGE to resolve the well-studied respiratory chain complexes in mitochondria of bovine heart muscle and Yarrowia lipolytica, as well as thylakoid localized complexes of Medicago sativa, Pisum sativum and Anabaena sp. PCC7120. Moreover, we determined the assembly/composition of the Anabaena sp. PCC7120 thylakoids and envelope membranes by HDN-PAGE. The analysis of isolated chloroplast envelope complexes by HDN-PAGE permitted us to resolve complexes such as the translocon of the outer envelope migrating at approximately 700 kDa or of the inner envelope of about 230 and 400 kDa with high resolution. By immunodecoration and mass spectrometry of these complexes we present new insights into the assembly/composition of these translocation machineries. The HDN-PAGE technique thus provides an important tool for future analyses of membrane complexes such as protein translocons.
    The Plant Journal 03/2011; 67(1):181-94. DOI:10.1111/j.1365-313X.2011.04577.x · 6.82 Impact Factor
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