Wayne State University

Methods

In-gel protein digestion (protocol from Bruker)

SYPRO Ruby gel staining (protocol from Invitrogen)

iTRAQ labeling of peptides (protocol from Applied Biosystems)

Enhanced Coomassie staining protocol: PMID: 19684561 or PMID: 20162584


 TIPS TO AVOID KERATIN CONTAMINATION WHEN PERFORMING SDS-PAGE

  • Keratin contamination is nearly impossible to completely avoid, and becomes more prominent when the proteins-of-interest are at low levels.
  • Always use NON-LATEX gloves and wear lab coats. 
  • Use the highest grade reagents possible (i.e. proteomics or mass spec grade). DTT, ß-mercaptoethanol and buffers are common sources of keratin.
  • Wash all glass plates thoroughly with 70% ethanol prior to casting an SDS-PAGE gel. 
  • After completing electrophoresis, disassemble in a laminar flow hood. 
  • Avoid storing gel in SARAN WRAP or similar material and instead use new, cleaned plastic or glass gel trays.
  • De-stain the gel in a clean container that has been rinsed thoroughly with 70% ethanol or methanol/acetonitrile.  
  • When excising gel slices, use fresh razor blades and nitrile gloves in a laminar flow hood.