1. Run SDS-PAGE or Native PAGE of protein samples,
2. Stain gel with Brilliant Blue R stain.
3. Wash gel with MilliQ water for several hours
4. Excise protein band(s), cut into ~1mm cubes and place in tubes
5. Add 500µL acetonitrile and incubate 10min until gel fragments become white.
6. Remove all liquid and add 50µL of 10mM DTT in 100mM ammonium bicarbonate and incubate 30min at 56°C.
7. Allow tubes to come to room temperature and add 500µL of acetonitrile, incubate 10min and remove all liquid.
8. Add 50µL of 55mM iodoacetamide in 100mM ammonium bicarbonate and incubate 20min at room temperature in the dark.
9. Add 500µL of acetonitrile and incubate until gel pieces turn white
10. Remove all liquid and cover gel fragments with 50µL of trypsin buffer, leave on ice for 30min.
11. Add an additional 50µL of trypsin buffer, leave on ice for 90min.
12. Add 20µL of 10mM ammonium bicarbonate, pH 8.5, and incubate at 37°C overnight.
13. Clean-up samples using Agilent Omix C18 pipette tips as per manufacturer instructions
14. Dry samples in speedvac
15. Re-suspend samples in 100µL of 0.1% formic acid just prior to analysis by mass spectrometry.
Brilliant Blue R stain: 60mg Brilliant blue R in 500mL H2O, 3mL of 6N HCl
Trypsin buffer: 13ng/uL trypsin in 10mM ammonium bicarbonate, pH 8.5, and 10% acetonitrile
NOTE: Take extra caution when handling gels and samples. Wear clean gloves to prevent/minimize keratin contamination.