- in Gel Digestion
- in Solution Digestion
- ZipTip
- Stage Tip protocols
- Coomassie Destaining
- SYPRO Destaining
- Metabolomics Sample Prep
BioID Blog
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.
(scroll up to see in-gel tryptic digestion protocol)
In-Solution Tryptic Digestion Protocol
- Download the In Solution Trypsin Digestion Protocol
- Protocol adapted from: Current Protocols in Protein Science, (1996-current) John Wiley and Sons, Inc. Brooklyn, Section 11.1.
- Notes: Read Promega's protocol for trypsin digestion (click on 'Protocol')
(scroll up to see in-solution tryptic digestion protocol)
Stage Tip Protocol
- Download the C18 Stage Tip (Stop and Go Extraction Tip) protocol for C18 peptide extraction/desalting
- Download the MCX Stage Tip protocol for "MCX-like" detergent and salt removal
(scroll up to see Stage Tip protocol)
ZipTip Protocol from CMSP, adapted from Millipore
- Download the ZipTip Protocol from CMSP
- Protocol can be accessed at Merk Millipore.
(scroll up to see ZipTip protocol)
Coomassie (CBB-R 250) Destaining Protocol
- Cut band from the gel
- 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.
- Wash gel piece with water.
- Shrink with 100% ACN (5 min sonicate)
- Reswell with water (5 min sonicate)
- Shrink with 100% ACN (5 min sonicate)
- Reswell with water (5 min sonicate)
- Wash with ACN:H2O = 1:1 (5 min sonicate) (steps 3 - 7: salt and other contaminant removal)
- Speedvac to near dryness.
- 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
(scroll up to see Coomassie destaining protocol)
SYPRO (Bio-Rad) Ruby Protein Gel Stain Removal
- After staining with Ruby Gel Stain excise protein band/spot from gel
- Wash the gel slices 2 x 25 mM ammonium bicarbonate in 50% acetonitrile and 1 x 100% acetonitrile
- Dry slices using a Speed Vac
- Reconstitute sample with proteolytic digestion buffer
SYPRO destaining notes provided by Bio-Rad Laboratories Technical Services department (Aug 2001
(scroll up to see Sypro Ruby gel stain removal protocol)