In-gel digest for Coomasie gels
- Before excising bands wash gels in MilliQ for 15’.
- Excise bands, cut as close to the bands as possible to minimize excess gel material, place in Eppendorf tubes.
- Add 10mM DTT (dilute DTT 1:10 in 50mM NH4HCO3) and incubate for 60‘ at 56°
- Remove DTT and add 50mM Iodacetamid (dilute Ioda 1:10 in NH4HCO3) and incubate for 60’ at RT
- Wash gel pieces with 50 – 100 µl MilliQ
- Wash gel pieces with 50 – 100 µl 50mM NH4HCO3 (30’). Pull off solution and dehydrate with 3/2 Acetonitrile/MilliQ.
- Repeat step 6. until gel pieces are colourless.
- Pull off solution and add pure Acetonitrile (10’) and air dry to complete dryness.
- Reswell gel pieces at 4°C on ice for 45’ in cold buffer containing trypsin and 50mM NH4HCO3 (10.0ng/µl) freshly prepared. The gel pieces should just be covered. Add more solution if pieces absorb all of solution.
- Pull off solution and discard, add the same buffer without trypsin (enough to cover gel pieces) and incubate overnight at 37°C.
- Collect supernatant, extract peptides by adding 3/2 Acentonitrile/0.1% TFA in MilliQ (60’) at room temperature.
- Collect elution, then cover gel with acetonitrile and incubate for 15’ at room temperature
- SpeedVac dry the combined washes/elutions