By Salvatore, Ed. Sechi
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Extra info for Quantitative Proteomics by Mass Spectrometry (Methods in Molecular Biology Vol 359)
3. Stable Isotope Labeling With ICAT Reagents 1. Eluted proteins from the p18 purifications (each containing approx 3 µg of total protein) are concentrated to 25 µL each in Nanosep 3 K centrifugal devices (see Note 6). 2. 3 and 1 mM EDTA. Keep an aliquot for analysis by silver-stained SDS-PAGE. 3. 3% and heating to 37°C (see Note 11). 4. The samples are reduced by addition of TBP to 5 mM and incubation at 37°C for 30 min. 05% SDS and 6 M urea. 5. One tube of heavy and one tube of light ICAT reagent are briefly spun in a microfuge to pellet the reagent.
4. Harvest of Cells, Lysis, and Estimation of Total Protein 1. Cells can be harvested using any standard protocol in tissue culture. In this example using HeLa cells, aspirate the medium from the cells and rinse the attached cells in warm phosphate buffered saline to remove remaining serum proteins. This is done twice to reduce the amount of serum albumin (a major component of serum) detectable in subsequent MS analyses. Scrape the cells in a small volume of lysis buffer (750 µL for a 14-cm dish), vortex frequently to ensure cell lysis, and obtain a small aliquot of each cell lysate for a Bradford assay to estimate protein concentration.
Monomeric avidin cartridge (Applied Biosystems) (see Note 3). 3. 4% trifluoroacetic acid (TFA). Prepare fresh. 4. Avidin wash buffer: 50 mM ammonium bicarbonate and 20% methanol. Prepare fresh. 5. Glass collection vials (Waters, Milford, MA; cat. no. WAT025054). Fig. 1 (continued from opposite page) the samples are prepared identically. Affinitypurified proteins from specific and control purifications are reduced, labeled with either the isotopically heavy or normal version of the ICAT reagent, and combined.
Quantitative Proteomics by Mass Spectrometry (Methods in Molecular Biology Vol 359) by Salvatore, Ed. Sechi