Dataset Information

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

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.

SUBMITTER: Kusch K

PROVIDER: S-EPMC5372224 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Publications

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

Kusch Kathrin K Uecker Marina M Liepold Thomas T Möbius Wiebke W Hoffmann Christian C Neumann Heinz H Werner Hauke B HB Jahn Olaf O

Proteomes 20170112 1

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 s ...[more]

PMID: 28248254

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Project description:Background informationThe discovery of green fluorescent protein (GFP) and its derivatives has revolutionized cell biology. These fluorescent proteins (FPs) have enabled the real-time observation of protein localization and dynamics within live cells. Applications of FP vary from monitoring gene/protein expression patterns, visualizing protein-protein interactions, measuring protein stability, assessing protein mobility, and creating biosensors. The utility of FPs also extends to biochemical approaches through immunoblotting and proteomic analyses, aided by anti-FP antibodies and nanobodies. FPs are notoriously robust proteins with a tightly folded domain that confers a strong stability and a relative resistance to degradation and denaturation.Methods and resultsIn this study, we report that various green, orange, and red FPs can be maintained in a native, fluorescent state during the entire process of protein sample extraction, incubation with sample buffer, loading, and migration on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) with only minor adaptations of traditional protocols. This protocol results in the ability to detect and quantify in-gel fluorescence (IGF) of endogenously-expressed proteins tagged with FPs directly after migration, using standard fluorescence-imaging devices. This approach eliminates the need for antibodies and chemiluminescent reagents, as well as the time-consuming steps inherent in immunoblotting such as transfer onto a membrane and antibody incubations.Conclusions and significanceOverall, IGF detection provides clearer data with less background interference, a sensitivity comparable to or better than antibody-based detection, a better quantification, and a broader dynamic range. After fluorescence imaging, gels can still be used for other applications such as total protein staining or immunoblotting if needed. It also expands possibilities by allowing the detection of FPs for which antibodies are not available. Our study explores the feasibility, limitations, and applications of IGF for detecting endogenously expressed proteins in cell extracts, providing insights into sample preparation, imaging conditions, and sensitivity optimizations, and potential applications such as co-immunoprecipitation experiments.

Dataset Information

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

Publications

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

Similar Datasets

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