How to prepare protein samples for nanoLC-MS/MS analysis?
Gel Samples—Short 1D PAGE: Load protein samples onto a 1D PAGE gel and electrophorese for only 1 cm (5-8 minutes at 200 V). After Coomassie staining, cut the entire stained region as one sample (2x5mm gel pieces) and send it for analysis. Long 1D PAGE: Load protein samples onto a 1D PAGE gel and electrophorese as usual. After Coomassie staining, cut the entire stained region into 5-10 smaller pieces (2x5mm in size). Place each piece in a separate tube and send for analysis. 2D PAGE: Load protein samples onto a 2D PAGE gel and electrophorese as usual. After Coomassie staining, cut out selected regions with multiple spots. Place each spot (2x5mm) in a separate tube and send for analysis. Note: Cut all gel samples into 2x5mm fragments. Solution Samples—Minimum Sample Amount: 5-50 micrograms. Avoid detergents, DMSO, glycerol, and other non-volatile solvents. Keep buffer concentrations to a minimum as high salt can interfere with ionization in MS. LC-ESI MS may be suitable for samples containing small amounts of salt or urea. However, optimal results are obtained in volatile solvents without detergents and with low buffer strength.
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