Sample Preparation and Gel Service
Sample preparation advice
1D SDS-PAGE gel analysis
1D and 2D gel imaging
Proteolytic digestion (In-gel or solution)
Samples can be received at the Facility internally from within the University, externally from other universities and from commercial companies. Samples are received in either a gel band or solution form (if samples are in solution they may require SDS PAGE analysis).
A series of washes is carried out using ammonium bicarbonate and acetonitrile to remove any excess salt and detergent. Samples are reduced with dithiothreitol breaking the cysteine disulphide bonds and then alkylated with iodoacetamide, stopping disulphide bonds reforming.
Samples are routinely digested with the proteolytic enzyme trypsin, for 4-18 hours @ 30° which cuts peptide bonds c-terminally at arginine and lysine, other enzymes can be used depending on the nature of experiment or sample.
The Facility runs two types of services either standard or enhanced. For the enhanced service samples are run on an OrbiTrap Velos mass spectrometer which is a faster and more sensitive instrument than that is used for the standard service. The standard service is recommended for gel bands whereas low intensity and complex samples such as gel sections and whole cell lysates are recommended for the enhanced service. Samples that require enhanced service need to pass our quality control before they can be analysed on the Mass spectrometer, this is carried out on a Thermo Scientific LTQ Velos. Failure is usually due to high levels of polymer or undigested material.
Raw mass spectrometer files are converted to MGF files which are then used to search relevant databases either using the Mascot search engine or Proteome Discoverer. Results are saved as Mascot reports or Excel spreadsheets and returned to the customer.