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Minimizing keratin contamination: SDS-PAGE Gel Handling

 
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02/10/2011

Minimizing keratin contamination: SDS-PAGE Gel Handling

  1. Gloves and clean lab coats should be worn at all times when working with gels and associated reagents, tubes, tips and apparatus.
  2. Whilst not essential if sufficient care is taken, the use of pre-cast gels can help reduce keratin contamination.
  3. Fresh staining reagents should be used to reduce the risk of contamination and prevent accumulation of contaminants.
  4. Use clean gel apparatus, staining trays and plastics (no dust!). Use new unopened boxes of tubes, tips etc. where possible., or make sure they have not been left open to air and/or plastics removed by ungloved hands. Do not use tubes from "community" containers.
  5. Most silver staining and Coomassie staining methods/kits are now compatible with mass spectrometry-based protein identification. Note: silver staining kits should not contain glutaraldehyde as a fixing agent. Most commercial kits will state if they are MS friendly. If you are in doubt contact proteomics support
  6. After staining, gels should be washed thoroughly in high-purity water prior to band excision. 2 x 15 minutes should be sufficient. Longer washes are suggested for thick gels (more than 1.5mm).
  7. For specific bands of interest, these should be cut from the gel with a clean, sharp razor blade. The band should be cut directly on the edge of the staining region; no borders of clear acrylamide should be left around the band.
  8. For GeLC experiments (i.e. ‘salami slicing’ of gel lanes) similarly always use a clean sharp blade (although obviously the instruction to avoid clear borders does not apply).
  9. Cut bands should be carefully placed into clean microcentrifuge tubes.
  10. The microcentrifuge tubes can be stored at -20 °C until they are ready for submission to the Bio-Synthesis Proteomics Centre for processing.
  11. A clear region of the gel, approximately the size of the gel band of interest, should be submitted with the sample(s). This will act as a blank control.
  12. Label the tubes well (initials, date and sample name)