Methods in Tau Cell Biology /
Methods in Tau Cell Biology, Volume 141, the latest release in the Methods in Cell Biology series, looks at methods involved in tau cell biology. Edited by leaders in the field, this volume provides proven, state-of-art techniques and relevant historical background and theory that aids researchers w...
| Άλλοι συγγραφείς: | , |
|---|---|
| Μορφή: | Ηλ. βιβλίο |
| Γλώσσα: | English |
| Έκδοση: |
Cambridge, MA :
Academic Press,
2017.
|
| Έκδοση: | First edition. |
| Σειρά: | Methods in cell biology ;
v. 141. |
| Θέματα: | |
| Διαθέσιμο Online: | Full Text via HEAL-Link |
Πίνακας περιεχομένων:
- Front Cover; Methods in Tau Cell Biology; Copyright; Contents; Contributors; Preface; Section I: Tau Structure, Oligomerization and Aggregation In Vitro; Chapter 1: Expression and isolation of recombinant tau; 1. Introduction; 2. Isolation of Untagged Tau Proteins; 2.1. Preparation of Tau-Expressing E. coli via Transformation; 2.2. (Alternative) Plate Cells From Previously Prepared Glycerol Stock of Transformed E. coli; 2.3. Overnight Starter Cultures; 2.4. Autoinduction of Tau Expression; 2.5. Harvest Bacteria; 2.6. Lyse by French Press; 2.7. Heat Denaturation and High-Speed Centrifugation
- 2.8. PC Chromatography2.8.1. Column precyclingaaDo this the same day you plan to use the column.; 2.8.2. Batch bind and run column; 2.9. HIC Column; 2.9.1. Equilibrate column 1 day in advance; 2.9.2. HIC column; 2.10. Protein Concentration and Buffer Exchange; 3. Isolation of His-Tagged Tau Proteins; 3.1. Modifications to Early Stages of the Protocol; 3.2. Affinity Chromatography With Ni-NTA Agarose; 3.3. Proteolytic Cleavage to Remove His-Tag; 3.4. Ni-NTA Column for Tag Removal; 4. Determination of Tau Concentration by SDS-PAGE and Comparison to a Tau Mass Standard; Acknowledgements
- 4.1.1.1. Fitting FCS curves4.2. smFRET Data Collection; 4.2.1. smFRET data analysis; 4.2.1.1. Thresholding; 4.2.1.2. Calculating ETeff; 4.2.1.3. Plotting data and analysis; 4.2.1.4. Calculating distances; 5. Summary; Acknowledgments; References; Chapter 3: Production of recombinant tau oligomers in vitro; 1. Introduction; 2. Purification of Recombinant Tau; 2.1. Growth and Induction of Proteins in E. coli; 2.1.1. Day 1: Transformation of E. coli; 2.1.2. Day 2: Initial Growth of E. coli Culture; 2.1.3. Day 3: Growth and Induction of Proteins in E. coli; 2.2. FPLC Purification of Tau Proteins
- 2.2.1. Day 1: Purification of Poly-His-Tagged Proteins and Size-Exclusion Chromatography2.2.2. Day 2: Anion Exchange Cleanup of Purified Tau; 2.3. Tau Protein Purification Buffers; 3. SDS-Lowry Protein Quantitation Assay Protocol; 3.1. Reagents; 4. Arachidonic Acid-Induced Formation of Tau Oligomers; 5. Sucrose Step Gradient Fractionation to Isolate Oligomers; 6. Electron Microscopy Imaging of Tau Oligomers; 6.1. Sample Preparation for TEM; 6.2. Materials; 7. Sandwich ELISA for Oligomer Detection; 7.1. TOC1 sELISA Protocol; 7.2. sELISA Quantification; 7.3. Materials; 8. Conclusion
