Native electrophoretic techniques to identify protein–protein interactions

Wittig, Ilka; Schägger, Hermann

doi:10.1002/pmic.200900151

Cited by 91 publications

(82 citation statements)

References 106 publications

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“…Blue native-PAGE (BN-PAGE) 3 has been successfully used to identify membrane protein-protein interactions and complexes (14). Mild neutral detergents are used for initial solubilization, and the anionic dye Coomassie Blue G-250 is used instead of SDS to introduce a negative charge to the protein complex for electrophoretic separation.…”

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Claudin-2 Forms Homodimers and Is a Component of a High Molecular Weight Protein Complex

Itallie

Mitic

Anderson

2011

Journal of Biological Chemistry

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Tight junctions are multiprotein complexes that form the fundamental physiologic and anatomic barrier between epithelial and endothelial cells, yet little information is available about their molecular organization. To begin to understand how the transmembrane proteins of the tight junction are organized into multiprotein complexes, we used blue native-PAGE (BN-PAGE) and cross-linking techniques to identify complexes extracted from MDCK II cells and mouse liver. In nonionic detergent extracts from MDCK II cells, the tight junction integral membrane protein claudin-2 was preferentially isolated as a homodimer, whereas claudin-4 was monomeric. Analysis of the interactions between chimeras of claudin-2 and -4 are consistent with the transmembrane domains of claudin-2 being responsible for dimerization, and mutational analysis followed by cross-linking indicated that the second transmembrane domains were arranged in close proximity in homodimers. BN-PAGE of mouse liver membrane identified a relatively discrete high molecular weight complex containing at least claudin-1, claudin-2, and occludin; the difference in the protein complex sizes between cultured cells and tissues may reflect differences in tight junction protein or lipid composition or post-translational modifications. Our results suggest that BN-PAGE may be a useful tool in understanding tight junction structure.

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Claudin-2 Forms Homodimers and Is a Component of a High Molecular Weight Protein Complex

Itallie

Mitic

Anderson

2011

Journal of Biological Chemistry

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“…co-workers as end point separation methods for characterization of solubilized mitochondrial membrane protein (super-)-complexes under close-to-native conditions (1)(2)(3). Subsequently, native gel electrophoresis became the method of choice for first dimension separation followed by second dimension SDS-PAGE in two-dimensional gel-based proteomic analyses (2D-BN) of membrane protein complexes.…”

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confidence: 99%

Cryo-slicing Blue Native-Mass Spectrometry (csBN-MS), a Novel Technology for High Resolution Complexome Profiling

Müller

Bildl

Haupt

et al. 2016

Molecular & Cellular Proteomics

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Blue native (BN)1 -PAGE and its colorless variant, colorless native PAGE, were originally developed by Schä gger and co-workers as end point separation methods for characterization of solubilized mitochondrial membrane protein (super-)-complexes under close-to-native conditions (1-3). Subsequently, native gel electrophoresis became the method of choice for first dimension separation followed by second dimension SDS-PAGE in two-dimensional gel-based proteomic analyses (2D-BN) of membrane protein complexes. After staining of the gel-separated proteins, protein spots are individually analyzed by different mass spectrometric methods, and the identified proteins were assigned to complexes based on their co-migration pattern (2D-BN-MS (4)). However, these 2D-BN-MS approaches exhibit the following severe shortcomings: (i) they are critically dependent on the staining properties of individual proteins; (ii) the size resolution of protein complexes is low; and (iii) the assignment of identified proteins to spots and complexes may be ambiguous. Therefore, application of 2D-BN-MS has remained largely restricted to the characterization of highly abundant and well defined membrane protein complexes such as complexes I-V of the respiratory chain in mitochondria (5-7), photosynthetic complexes (8 -10), or viruses (11).In a first attempt to overcome these shortcomings of 2D-BN-MS, Wessels et al. (12) coupled BN-PAGE separation more directly to MS analysis by manually cutting the gel lane into 24 slices/sections of about 2 mm width that were separately digested and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Their study on HEK cell mitochondria identified 59 of the 90 canonical subunits of the oxidative respiratory chain (OXPHOS) complexes I-V. The respective protein abundance profiles (based on standard label-free quantification) showed clustering of their peak maxima into the expected complexes I-V. Since then, this onedimensional BN-MS methodology has been gradually improved with respect to quality of the native gel separation, LC-MS/MS sensitivity, and robustness of the quantitative evaluation. Thus, two recent studies on human mitochondrial preparations (each analyzing two BN separations in 60 and 24 slices, respectively) reported identification and hierarchical profile clustering of 464 (13)

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“…CN-PAGE separation relies on both size and charge (61), but BN-PAGE can separate protein complexes according to size only due to the presence of anionic Coomassie Blue dye at neutral pH (56,61). As in CN-PAGE, we could detect both A and B forms of NEMO by BN-PAGE analysis using VP16-stimulated NEMO-WT cell extracts, and the overall amount of the B form was much smaller than the A form in BN-PAGE compared with that found in CN-PAGE (Fig.…”

Section: Ipo3 Directly and Inducibly Interacts With Nemo-wt But Not mentioning

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IPO3-mediated Nonclassical Nuclear Import of NF-κB Essential Modulator (NEMO) Drives DNA Damage-dependent NF-κB Activation

Hwang

McCool²,

Wan

et al. 2015

Journal of Biological Chemistry

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Background: Nuclear import of NEMO is critical for DNA damage-dependent NF-B signaling, but the mechanism remains unknown. Results: IPO3 binds NEMO, promotes its nuclear import, and is critical for DNA damage-dependent NF-B activation. Conclusion: IPO3 is a nonclassical nuclear import receptor for NEMO. Significance: IPO3 is a new player in DNA damage-dependent NF-B signaling with implications in cancer therapy.

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Native electrophoretic techniques to identify protein–protein interactions

Cited by 91 publications

References 106 publications

Claudin-2 Forms Homodimers and Is a Component of a High Molecular Weight Protein Complex

Claudin-2 Forms Homodimers and Is a Component of a High Molecular Weight Protein Complex

Cryo-slicing Blue Native-Mass Spectrometry (csBN-MS), a Novel Technology for High Resolution Complexome Profiling

IPO3-mediated Nonclassical Nuclear Import of NF-κB Essential Modulator (NEMO) Drives DNA Damage-dependent NF-κB Activation

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