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

Ludwig Maximilian University, Munich, Germany.
Proteomics (Impact Factor: 3.81). 09/2007; 7 Suppl 1(S1):6-16. DOI: 10.1002/pmic.200700205
Source: PubMed


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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Available from: Lutz A Eichacker,
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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.
    Journal of plant physiology 06/2014; 171(15):1423-1435. DOI:10.1016/j.jplph.2014.06.006 · 2.56 Impact Factor
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    • "However, due to the limitation of the resolution of electrophoresis system, more than one protein complex could be separated to the same molecular mass band during BN-PAGE and more than one protein subunit might be resolved to have same molecular mass. In other word, should not all protein subunits in the same vertical row do belong to same protein complex [22]. In our study, 34 unique protein complexes were identified, of which 33 were known protein complexes. "
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    ABSTRACT: Placental malfunction induces pregnancy disorders which contribute to life-threatening complications for both the mother and the fetus. Identification and characterization of placental multi-protein complexes is an important step to integratedly understand the protein-protein interaction networks in placenta which determine placental function. In this study, blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (BN/SDS-PAGE) and Liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to screen the multi-protein complexes in placenta. 733 unique proteins and 34 known and novel heterooligomeric multi-protein complexes including mitochondrial respiratory chain complexes, integrin complexes, proteasome complexes, histone complex, and heat shock protein complexes were identified. A novel protein complex, which involves clathrin and small conductance calcium-activated potassium (SK) channel protein 2, was identified and validated by antibody based gel shift assay, co-immunoprecipitation and immunofluorescence staining. These results suggest that BN/SDS-PAGE, when integrated with LC-MS/MS, is a very powerful and versatile tool for the investigation of placental protein complexes. This work paves the way for deeper functional characterization of the placental protein complexes associated with pregnancy disorders.
    PLoS ONE 04/2013; 8(4):e62988. DOI:10.1371/journal.pone.0062988 · 3.23 Impact Factor
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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 · 0.36 Impact Factor
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