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Proteomes 2017, 5(1), 3; doi:10.3390/proteomes5010003

Partial Immunoblotting of 2D-Gels: A Novel Method to Identify Post-Translationally Modified Proteins Exemplified for the Myelin Acetylome

1
Department of Neurogenetics, Max-Planck-Institute of Experimental Medicine, 37075 Göttingen, Germany
2
Proteomics Group, Max-Planck-Institute of Experimental Medicine, 37075 Göttingen, Germany
3
Center for Nanoscale Microscopy and Molecular Physiology of the Brain, 37075 Göttingen, Germany
4
Free Floater (Junior) Research Group Applied Synthetic Biology, Georg-August University Göttingen, Institute for Microbiology and Genetics, 37077 Göttingen, Germany
Present address: Accurion GmbH, 37079 Göttingen, Germany.
Present address: Applied Synthetic Biology Group, Max-Planck-Institute of Molecular Physiology, 44227 Dortmund, Germany.
*
Author to whom correspondence should be addressed.
Academic Editors: Jens R. Coorssen, Alfred L. Yergey and Jacek R. Wisniewski
Received: 29 September 2016 / Revised: 23 December 2016 / Accepted: 4 January 2017 / Published: 12 January 2017
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Abstract

Post-translational modifications (PTMs) play a key role in regulating protein function, yet their identification is technically demanding. Here, we present a straightforward workflow to systematically identify post-translationally modified proteins based on two-dimensional gel electrophoresis. Upon colloidal Coomassie staining the proteins are partially transferred, and the investigated PTMs are immunodetected. This strategy allows tracking back the immunopositive antigens to the corresponding spots on the original gel, from which they are excised and mass spectrometrically identified. Candidate proteins are validated on the same membrane by immunodetection using a second fluorescence channel. We exemplify the power of partial immunoblotting with the identification of lysine-acetylated proteins in myelin, the oligodendroglial membrane that insulates neuronal axons. The excellent consistency of the detected fluorescence signals at all levels allows the differential comparison of PTMs across multiple conditions. Beyond PTM screening, our multi-level workflow can be readily adapted to clinical applications such as identifying auto-immune antigens or host-pathogen interactions. View Full-Text
Keywords: post-translational modification (PTM); 2D gel electrophoresis (2DE) blot; myelin; acetylome; SERPA; immunoproteomics; tubulin acetylation; cyclic nucleotide phosphodiesterase (CNP); septin 8 (SEPT8); isoelectric focusing (IEF); immunoblot; cryo immuno-electron microscopy (IEM) post-translational modification (PTM); 2D gel electrophoresis (2DE) blot; myelin; acetylome; SERPA; immunoproteomics; tubulin acetylation; cyclic nucleotide phosphodiesterase (CNP); septin 8 (SEPT8); isoelectric focusing (IEF); immunoblot; cryo immuno-electron microscopy (IEM)
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Kusch, K.; Uecker, M.; Liepold, T.; Möbius, W.; Hoffmann, C.; Neumann, H.; Werner, H.B.; Jahn, O. Partial Immunoblotting of 2D-Gels: A Novel Method to Identify Post-Translationally Modified Proteins Exemplified for the Myelin Acetylome. Proteomes 2017, 5, 3.

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