Dissolve 0.37 g Urea in 700 100 mM Tris-HCl, pH 8.5. Make up fresh each time. μl Reducing 0.5 M TCEP in Trypsin digestion kit, Rm 2-27, 4°C; dilute 10-1 with water for 50 mM conc. Alkylating agent - 500 mM Iodoacetamide (IAA) Dissolve 0.092 g in 1 ml HPLC water. Make up fresh in amber 1.5 ml tube. Trypsin 1 Trypsin gold stock in -80°C freezer – μg/μl Digestion Buffer (W1) 10 mg of ammonium bicarbonate + 5 ml ultrapure water (final conc 2 mg/ml = – 25 mM). Store at 4°C for up to 3 months but always check pH with pH paper before use to make sure it is at pH 8. Trypsin digestion will not work in acidic solution. Buffer A 0.1% formic acid in HPLC-grade water (vol/vol) – Buffer B 0.1% formic acid in acetonitrile (ACN) –
Dissolve 0.37 g Urea in 700 μl 100 mM Tris-HCl, pH 8.5. Make up fresh each time. Reducing 0.5 M TCEP in Trypsin digestion kit, Rm 2-27, 4°C; dilute 10-1 with water for 50 mM conc. Alkylating agent - 500 mM Iodoacetamide (IAA) Dissolve 0.092 g in 1 ml HPLC water. Make up fresh in amber 1.5 ml tube. Trypsin – 1 μg/μl Trypsin gold stock in -80°C freezer Digestion Buffer (W1) – 10 mg of ammonium bicarbonate + 5 ml ultrapure water (final conc 2 mg/ml = 25 mM). Store at 4°C for up to 3 months but always check pH with pH paper before use to make sure it is at pH 8. Trypsin digestion will not work in acidic solution. Buffer A – 0.1% formic acid in HPLC-grade water (vol/vol) Buffer B – 0.1% formic acid in acetonitrile (ACN)
trypsin-gold-in-soln-digest-protocol_mfb.docx