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Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins
- Source :
- Journal of visualized experiments : JoVE, Journal of visualized experiments : JoVE, JoVE, 2016, ⟨10.3791/55001⟩, Journal of visualized experiments : JoVE, 2016, 118, ⟨10.3791/55001⟩
- Publication Year :
- 2016
- Publisher :
- HAL CCSD, 2016.
-
Abstract
- Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed. 118
- Subjects :
- 0301 basic medicine
Magnetic Resonance Spectroscopy
General Chemical Engineering
Tau protein
tau Proteins
Intrinsically disordered proteins
Biochemistry
General Biochemistry, Genetics and Molecular Biology
Isotopic labeling
03 medical and health sciences
Alzheimer Disease
Protein purification
mental disorders
Triple-resonance nuclear magnetic resonance spectroscopy
Humans
Protein phosphorylation
Phosphorylation
[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM]
ComputingMilieux_MISCELLANEOUS
biology
General Immunology and Microbiology
Chemistry
General Neuroscience
Nuclear magnetic resonance spectroscopy
Recombinant Proteins
3. Good health
[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]
Intrinsically Disordered Proteins
030104 developmental biology
biology.protein
Issue 118
nuclear magnetic resonance spectroscopy
isotopic labeling
intrinsically disordered protein
recombinantprotein
protein purification
protein phosphorylation
NMR data acquisition
resonance assignment
Subjects
Details
- Language :
- English
- ISSN :
- 1940087X
- Database :
- OpenAIRE
- Journal :
- Journal of visualized experiments : JoVE, Journal of visualized experiments : JoVE, JoVE, 2016, ⟨10.3791/55001⟩, Journal of visualized experiments : JoVE, 2016, 118, ⟨10.3791/55001⟩
- Accession number :
- edsair.doi.dedup.....36b699a589568f5f62ed8a40f27010c3