Amicon ultra 15 ultracel 10 kd

Cells were lysed in ten volumes of modified SDT buffer (0.1 M Tris-HCL, pH 7.6, 0.1 M DTT, 1% SDS, 1% SDC) and incubated at 95 °C for 5 min. The lysate was sonicated to shear genomic DNA, and clarified by centrifugation at 20,000 × g for 15 min at 20 °C. The supernatant was transferred to ultrafiltration units (Millipore, Amicon Ultra 15 Ultracel 10 KD) and centrifuged at 4000 × g for 40 min. After centrifugation, the concentrates were mixed with 2 mL of 50 mM iodoacetamide in UA solution (8 M urea, 100 mM Tris-HCl pH 8.5) and incubated in darkness at room temperature (RT) for 30 min followed by centrifugation for 30 min. After alkylation, the filter units were washed four times with 10 ml UA buffer and two times 10 mL of 50 mM ammonium bicarbonate by centrifugation at 4000 × g. Proteins were then digested with Lys-C (1:100, w/w, Wako) for 6 h at 37 °C and trypsin (1:50, w/w, Promega) overnight at 37 °C. The resulting peptide mixture was acidified (pH 2.0) with formic acid, loaded onto Sep-Pak tC18 cartridges (Waters), desalted and eluted with 70% acetonitrile. The eluted peptides were lyophilized and stored at −80 °C before analysis.

Cells were lysed in ten volumes of modified SDT buffer (0.1 M Tris HCL, pH 8.5, 0.1 M DTT, 1% SDS, 1% SDC) and incubated at 95°C for 5 mins. The lysate was sonicated to shear genomic DNA, and clarified by centrifugation at 20,000 g for 15 min at 20°C. The supernatant was transferred to ultrafiltration units (Millipore, Amicon Ultra 15 Ultracel 10 KD) and centrifuged at 4,000 g for 40 mins. After centrifugation, the concentrates were mixed with 2 mL of 50 mM iodoacetamide in UA solution (8 M urea, 100 mM Tris-HCl pH 8.5) and incubated in darkness at room temperature (RT) for 30 mins followed by centrifugation for 30 mins. After alkylation, the filter units were washed four times with 10 ml UA buffer and two times 10 mL of 50 mM ammonium bicarbonate by centrifugation at 4,000 g. Proteins were then digested with Lys-C (1:100, w/w, Wako) for 6 hrs at 37°C and trypsin (1:50, w/w, Promega) overnight at 37°C. The resulting peptide mixture was acidified (pH 2) with formic acid, loaded onto Sep-Pak tC18 cartridges (Waters), desalted and eluted with 70% acetonitrile. The eluted peptides were lyophilized and stored at -80°C before analysis.