Download Analysis of Post-Translational Modifications and Proteolysis by Jennifer Elizabeth Grant, Hong Li PDF

By Jennifer Elizabeth Grant, Hong Li

This quantity highlights proteomics stories of quantitative PTM adjustments in either peripheral and critical frightened method proteomes using the latest advances in mass spectrometry. Chapters contain sensible details bearing on the basics of pattern practise, liquid chromatography, and tandem mass spectrometry instrumental research and may elucidate top practices within the interpretation of knowledge utilizing smooth bioinformatics techniques. Written for the preferred Neuromethods series, chapters contain the type of aspect and key implementation recommendation that guarantees winning leads to the laboratory.

Authoritative and practical, Analysis of Post-Translational ameliorations and Proteolysis in Neuroscience aims to make sure winning ends up in the extra learn of this important field.

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Analysis of Post-Translational Modifications and Proteolysis in Neuroscience

This quantity highlights proteomics reviews of quantitative PTM alterations in either peripheral and relevant anxious process proteomes using the newest advances in mass spectrometry. Chapters comprise sensible info referring to the basics of pattern training, liquid chromatography, and tandem mass spectrometry instrumental research and should elucidate most sensible practices within the interpretation of information utilizing glossy bioinformatics methods.

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Additional resources for Analysis of Post-Translational Modifications and Proteolysis in Neuroscience

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5 % Acetic acid in water 7. À80  C freezer (Thermo) 8. Speed-vacuum (Labconco) 34 Haopeng Xiao et al. 5 Boronic Acid Enrichment 1. Boronic acid (BA) conjugated magnetic beads stored in ethanol (BA concentration ¼ 6 mM) 2. 0) 200 mM ammonium acetate buffer 3. Elution buffer: ACN:H2O:TFA ¼ 50:49:1 4. Incubating shaker 5. Magnetic rack 6. À80  C freezer 7. 6 PNGase F Treatment 1. PNGase F (Sigma) 2. ) 3. 7 Glycopeptide Fractionation 1. 4) 2. HPLC (Agilent) 3. 6 Â 250 mm 5 μm particle reversed phase column (Waters) 4.

Freeze the eluate in a À80  C freezer for 15 min 10. Lyophilize the sample using a speed-vacuum sample dry system. 5 Boronic Acid Enrichment 1. For every 5 mg of peptides, pick up 250 μL of BA magnetic bead slurry to work with (Note 4) 2. 0 (binding buffer) 3. Resuspend BA beads in 500 μL of binding buffer, then transfer the slurry into the Eppendorf tube containing all peptides 4. Incubate the tube at 37  C for 1 h with appropriate shaking 5. Wash beads five times with 500 μL binding buffer 6.

1 33 Materials Cell Culture 1. Human HeLa or HEK293 cells (or any other types of mammalian cells) 2. Culture medium: Dulbecco’s Modified Eagle Medium (DMEM), low glucose (1 g/L), with 10 % fetal bovine serum (FBS). 3. 2 Protein Extraction 1. 4 2. 1 pellet/mL protease inhibitor EDTA-free (Roche) 3. Ice bath 4. Refrigerated benchtop centrifuge (Thermo) 5. , 15 and 50 mL centrifuge tubes, pipettes, Eppendorf tubes, etc. 3 Protein Reduction, Alkylation, Precipitation, and InSolution Digestion 1. 1 M dithiothreitol (DTT) 2.

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