The gel spots were dehydrated in 50ul 100% ACN for 20 min at room temperature and rehydrated in 25 mM NH4HCO3 containing 0.005 ug/ul modified trypsin (Promega).
Destained gel spots were dehydrated by rinsing twice with 100% ACN.
These spots were then dehydrated twice for 10 minutes vortexing with 200 ul of 25 mM ammonium bicarbonate/50% acetonitrile (ACN).
Protein spots were then dehydrated using 100% acetonitrile for 10 min and dried in a vacuum SpeedVac.
In short, protein spots were excised, destained, dehydrated, and digested overnight with modified Trypsin (Roche Diagnostics, Mannheim, Germany).
For protein identification, excised gel spots were washed and dehydrated with acetonitrile (Rathburn, Scotland, HPLC grade S).
If one spot on the food is colder than the other, the water molecules will sublimate, migrate and form ice crystals on the coldest spot, leaving the other parts dehydrated.
If one spot on the food is colder than the other, the water molecules will sublimate, migrate and form ice crystals on the coldest spot, leaving the other parts dehydrated., the water molecules in it form into ice crystals.
Fixed biomass (10 μl) was spotted on Teflon-coated microscopic slides and dehydrated for 3 min in subsequently 50%, 80%, and 96% ethanol.
Spots were excised from the 2D gels using a manual spot picker (Genetix), washed with 0.1 M ammonium bicarbonate and dehydrated with acetonitrile.
Before digestion, spots were washed twice in Milli-Q water and dehydrated with acetonitrile, then reswelled on ice for about 45 min in 5 μL 25 mM NH4HCO3 digestion buffer containing 12.5 ng/μL porcine trypsin (Promega).
