By Jennifer Elizabeth Grant, Hong Li
This quantity highlights proteomics reviews of quantitative PTM alterations in either peripheral and valuable anxious method proteomes using the latest advances in mass spectrometry. Chapters comprise functional details concerning the basics of pattern practise, liquid chromatography, and tandem mass spectrometry instrumental research and may elucidate top practices within the interpretation of knowledge utilizing glossy bioinformatics ways. Written for the preferred Neuromethods series, chapters contain the type of element and key implementation suggestion that guarantees profitable ends up in the laboratory.
Authoritative and practical, Analysis of Post-Translational alterations and Proteolysis in Neuroscience aims to be certain profitable ends up in the extra learn of this very important field.
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Extra resources for Analysis of Post-Translational Modifications and Proteolysis in Neuroscience
Clear solution by centrifugation for 5 min at 10,000 rcf (g) at 4 C in a microcentrifuge. The insoluble pellet may appear considerable. This will not pose a problem since most of the peptide will be soluble. Cool on ice. 3. Wash motif antibody-bead slurry sequentially, four times with 1 mL of 1Â PBS, and resuspend as a 50 % slurry in PBS to remove the glycerol contaminating buffer. 4. Transfer the peptide solution into the microfuge tube containing motif antibody beads. Pipet sample directly on top of the beads at the bottom of the tube to ensure immediate mixing.
The maximum capacity is 5 % cartridge weight 3. 5 % acetic acid 4. 1 % TFA 5. Load sample 6. 1 % TFA 7. 5 % acetic acid to remove TFA 8. 5 % acetic acid in water 9. 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.
2 LysC Digestion 1. 0. Aliquot for single use and store at À80 C. 16 Hongbo Gu et al. 2. Add LysC solution to peptides at 1:250 (w:w). For 20 mg sample, use 20 mg Ä 250 ¼ 80 μg Â 1 μL/5 μg ¼ 16 μL LysC and digest overnight at room temperature. 3. Analyze the lysate before and after digest by SDS-PAGE to check for complete digestion. 4. 12). 7 Sep-Pak® C18 Purification of Lysate Peptides 1. 7 mL Sep-Pak columns from Waters Corporation, WAT051910. 2. NOTE: Sep-Pak® C18 purification utilizes reversed-phase (hydrophobic) solid-phase extraction.
Analysis of Post-Translational Modifications and Proteolysis in Neuroscience by Jennifer Elizabeth Grant, Hong Li