By Jörg Reinders
This quantity provides an outline of up to date quantitative proteomics tools besides directions on facts interpretation, whereas delivering examples on how you can enforce proteomics into platforms biology. Written within the hugely profitable Methods in Molecular Biology series layout, chapters contain introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, effectively reproducible laboratory protocols, and tips about troubleshooting and warding off identified pitfalls.
Thorough and sensible, Proteomics in platforms Biology: equipment and Protocols is a precious source for researchers who're attracted to utilizing proteomics suggestions to aid resolution organic and scientific questions.
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Additional info for Proteomics in Systems Biology: Methods and Protocols
Example text
Sonication water bath. 4 Peptide Separation by Strong Cation-Exchange HPLC 1. HPLC instrument (Hewlett Packard 1100 series) with autosampler (Agilent Technologies 1200). 2. 1 mm—300 Å; Hichrom Ltd, UK). 3. Guard cartridge (Hichrom Ltd, UK; see Note 9). 4. 75), 25 % acetonitrile (see Note 10). 5. 75), 25 % acetonitrile (see Note 10). 6. Vacuum centrifuge (Eppendorf, UK). 6 Sample Cleanup Prior to Mass Spectrometry 1. 3100 OFFGEL Fractionator (Agilent Technologies, UK). 2. OFFGEL High Resolution Kit, pH 3–10, 12 samples, 24 fractions (Agilent Technologies, UK).
Add appropriate amount of beads to the peptides and incubate the peptide-bead suspension for 10 min on a vortex mixer at low speed. Afterwards, centrifuge at 3000 rcf for 1 min to pellet the beads. Collect the supernatant in a LoBind Eppendorf tube. 5. Repeat the enrichment process in a stepwise manner (Fig. 2) by treating the supernatant with freshly prepared suspension of Fig. 2 Serial enrichment of phosphopeptides from an iTRAQ-labeled sample using TiO2 beads. It is recommendable to perform a second round of enrichment (from Step 2) to enhance the phosphoproteome coverage.
C) Sample loading: Place the sample on the material and reload the flow through three times. 1 % TFA. 1 % TFA 2. , 2 μL) of eluate from each sample and dry it completely in the SpeedVac. Snap freeze the remaining volume using liquid nitrogen and store the frozen eluates at −80 °C until further use. See Note 8. 3. Measure the samples (2 μL) using nano-LC with UV detection (214 nm) or LC-MS. If they look reproducible, take 100 μg aliquots from each sample, dry them in the SpeedVac, and proceed with iTRAQ labeling.