Enzymatic digestion for sequencing

 

Enzymes :   Trypsine, Chymotrypsine, Endoproteinases e.g. Lys-C,..

1.                dry protein in Eppendorf +

                   25 µl 8M urea

                   0.4 M NH4HCO3

                   c of protein > 20 µg / ml

2.                pH at 7.5 - 8.5 (pH-Papier)

3.                remove 10 % for total hydrolysis und Aa - analysis

4.                + 5 µl 45 mM dithiothreitol

5.                15 min 50°C, cool

6.                + 5 µl 100 mM iodoacetamide

7.                15 min RT

8.                + 60 µl water

9.                + 1:25 enzyme / protein w/w in 5 µl

                   Note !!  Even traces (0.0005 %) of SDS are critical

10.              24 h 37°C

11.              Stop reaction by injection in HPLC or

                   frieze at < -18°C

 

Enzyme :     Sequencing Grade e.g. Roche as stock solution of 1 mg/ml ( -20°C)