Crude and high abundant protein depleted plasma and serum samples were separated in two dimensions using the GE Life Sciences Ettan DIGE system protocol. Briefly, each sample (50 μg) was minimally labeled with 1 μl of 200 pM Cy2, Cy3 or Cy5 for 30 minutes. Labeling reactions were stopped by the addition of 1 μl of 1 mM lysine. The samples were pooled together and added to rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1.2% DeStreak, 1% pharmalytes). A final volume of 450 μl sample was loaded onto 24 cm pH 3–10NL Immobiline DryStrips (GE Life Sciences) and focused by active overnight rehydration, followed by isoelectric focusing for a total of 62,500 Vhrs. Strips were equilibrated in SDS equilibration buffer (6 M urea, 30% glycerol, 2% SDS) for 15 min with 10 mg/ml DTT, then 15 min in fresh buffer with 25 mg/ml 15 min with IAA, then applied to DIGE gels (GE Life Sciences) for 2nd dimension separation. The resulting CyDye labeled protein gels were scanned using 100 micron resolution on Typhoon 9410 (GE Life Sciences).
Dige gels
Manufactured by GE Healthcare
DIGE gels are a type of electrophoresis gel used in proteomic research. They enable the simultaneous separation and comparison of multiple protein samples on a single gel, facilitating the identification of differentially expressed proteins.
Automatically generated - may contain errors
2 protocols using dige gels
1
Proteomic analysis of plasma and serum
2
Two-Dimensional Proteomic Profiling
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Crude and high abundant protein depleted effluent and serum samples were separated in 2 dimensions according to GE Life Sciences Ettan DIGE system protocol. Briefly, each sample (50 μg) was minimally labeled with 1 μl of 200 pM Cy2, Cy3 or Cy5 for 30 min. Labeling reactions were stopped by the addition of 1 μl of 1 mM lysine. The samples were pooled together and added to rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1.2% DeStreak, 1% pharmalytes). A final volume of 450 μl sample was loaded onto 24 cm ph3-10NL Immobiline DryStrips (GE Life Sciences) and focused by active overnight rehydration, followed by isoelectric focusing for a total of 62,500 Vhrs. Strips were equilibrated in SDS equilibration buffer (6 M urea, 30% glycerol, 2% SDS) for 15 min with 10 mg/ml DTT, then 15 min in fresh buffer with 25 mg/ml 15 min with IAA, then applied to DIGE gels (GE Life Sciences) for 2nd dimension separation. The resulting CyDye labeled protein gels were scanned using 100 micron resolution on Typhoon 9410 (GE Life Sciences).
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