date: 2017-01-12T04:23:33Z
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pdf:docinfo:title: Partial Immunoblotting of 2D-Gels: A Novel Method to Identify Post-Translationally Modified Proteins Exemplified for the Myelin Acetylome
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subject: 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.
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dc:title: Partial Immunoblotting of 2D-Gels: A Novel Method to Identify Post-Translationally Modified Proteins Exemplified for the Myelin Acetylome
modified: 2017-01-12T04:23:33Z
cp:subject: 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.
pdf:docinfo:subject: 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.
pdf:docinfo:creator: Kathrin Kusch, Marina Uecker, Thomas Liepold, Wiebke Möbius, Christian Hoffmann, Heinz Neumann, Hauke B. Werner and Olaf Jahn
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Author: Kathrin Kusch, Marina Uecker, Thomas Liepold, Wiebke Möbius, Christian Hoffmann, Heinz Neumann, Hauke B. Werner and Olaf Jahn
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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)
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dc:creator: Kathrin Kusch, Marina Uecker, Thomas Liepold, Wiebke Möbius, Christian Hoffmann, Heinz Neumann, Hauke B. Werner and Olaf Jahn
dcterms:created: 2017-01-12T04:23:33Z
Last-Modified: 2017-01-12T04:23:33Z
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title: Partial Immunoblotting of 2D-Gels: A Novel Method to Identify Post-Translationally Modified Proteins Exemplified for the Myelin Acetylome
Last-Save-Date: 2017-01-12T04:23:33Z
pdf:docinfo: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)
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creator: Kathrin Kusch, Marina Uecker, Thomas Liepold, Wiebke Möbius, Christian Hoffmann, Heinz Neumann, Hauke B. Werner and Olaf Jahn
dc:subject: 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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meta:keyword: 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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pdf:docinfo:created: 2017-01-12T04:23:33Z