Sep pak vac rc cartridge

Example 41

About 1 mg protein in 9 M urea lysis buffer was reduced by the addition of 1/10 volume of 55 mM DTT to the cleared cell supernatant and incubated for 30 min at room temperature, followed by addition of 120 mM iodoacetamide at a volume equal to the DTT solution and incubated for 30 min at room temperature in the dark. The samples were then diluted 1.5-fold using 20 mM Hepes (pH8) to a final concentration of 6 M urea. LysC (Wako 129-02451) was added with a LysC to protein ratio of 1:100 and incubated at 37° C. overnight. The samples were further diluted to a final concentration of ˜1 M urea using 20 mM Hepes (pH8). Sequencing-grade trypsin (Promega) was added with a trypsin to protein ratio of 1:50 (w/w). After 4 hours of digestion at 37° C., the peptide solution was cleaned up using a C18 cartridge (Waters Sep-Pak Vac RC Cartridge, 100 mg Sorbent per Cartridge). The amount of peptide obtained was measured using Nanodrop (ThermoScientific).

Example 41

About 1 mg protein in 9 M urea lysis buffer was reduced by the addition of 1/10 volume of 55 mM DTT to the cleared cell supernatant and incubated for 30 min at room temperature, followed by addition of 120 mM iodoacetamide at a volume equal to the DTT solution and incubated for 30 min at room temperature in the dark. The samples were then diluted 1.5-fold using 20 mM Hepes (pH8) to a final concentration of 6 M urea. LysC (Wako 129-02451) was added with a LysC to protein ratio of 1:100 and incubated at 37° C. overnight. The samples were further diluted to a final concentration of ˜1 M urea using 20 mM Hepes (pH8). Sequencing-grade trypsin (Promega) was added with a trypsin to protein ratio of 1:50 (w/w). After 4 hours of digestion at 37° C., the peptide solution was cleaned up using a C18 cartridge (Waters Sep-Pak Vac R C Cartridge, 100 mg Sorbent per Cartridge). The amount of peptide obtained was measured using Nanodrop (ThermoScientific).