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Protocols

EMBL Proteomics Core Facility Sample Preparation Guidelines

Read important and thorough accounts of sample prep-related details

See: User Guide, DO's AND DONT's and FAQs

In-Gel Tryptic Digestion Protocol

  • Download the In Gel Trypsin Proteolysis Protocol
  • Protocol adapted from: Shevchenko, Andrej; Wilm, Matthias; Vorm, Ole; Mann, Matthias. Mass spectrometric sequencing of proteins from silver-stained polyacrylaminde gels, Analytical Chemistry, 68: 850-858 (1996).
  • For Review on gel-separated proteins see:
  • Patterson, S. D., Reudi Aebersold (1995) Mass spectrometric approaches for the identification of gel-separated proteins. Electrophoresis, 16: 1791-1814.

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In-Solution Tryptic Digestion Protocol

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Stage Tip Protocol

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ZipTip Protocol from MSP, adapted from Millipore

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Coomassie (CBB-R 250) Destaining Protocol
  1. Cut band from the gel
  2. Wash several times with 10 mM dithiotreitol (DTT), 0.1 M 4-ethylmorpholine acetate (pH 8.1) in 50% acetonitrile (ACN); (3 minutes in microwave oven per wash is suggested). Repeat until the Coomassie is completely removed.
  3. Wash gel piece with water.
  4. Shrink with 100% ACN (5 min sonicate)
  5. Reswell with water (5 min sonicate)
  6. Shrink with 100% ACN (5 min sonicate)
  7. Reswell with water (5 min sonicate)
  8. Wash with ACN:H2O = 1:1 (5 min sonicate) (steps 3 - 7: salt and other contaminant removal)
  9. Speedvac to near dryness.
  10. Reconstitute sample with proteolytic digestion buffer→proteolytic digest

Reference: Karel Drbal, Pavla Angelisova, Ivan Hilgert, Jan Cerny, Petr Novak, and Vaclav Horejsi(2001) A proteolytically truncated form of free CD18, the common chain of leukocyte integrins, as a novel marker of activated myeloid cells. Blood, 98(5): 1561-6, with modifications by Petr Novak at the Laboratory of Molecular Structure Characterization, Institute of Microbiology, Prague

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SYPRO (Bio-Rad) Ruby Protein Gel Stain Removal
  1. After staining with Ruby Gel Stain excise protein band/spot from gel
  2. Wash the gel slices 2 x 25 mM ammonium bicarbonate in 50% acetonitrile and 1 x 100% acetonitrile
  3. Dry slices using a Speed Vac
  4. Reconstitute sample with proteolytic digestion buffer

SYPRO destaining notes provided by Bio-Rad Laboratories Technical Services department (Aug 2001

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