In-gel protein digestion (protocol from Bruker)
SYPRO Ruby gel staining (protocol from Invitrogen)
iTRAQ labeling of peptides (protocol from Applied Biosystems)
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.