Enzymatic digestion within gel

 

Enzymes :      Trypsin, Chymotrypsin, Endoproteinases,....

Gel :               <= 0.5 x 12 cm piece

1.                    Extract gel slice with 95% acetone

                       30 min 4°C, remove acetone but keep it!

                       dry in Speed-Vac

2.a                  Gel slice +

                       500 µl 0.1 M NH4HCO3

                       (negative control !)

                       > 4 h RT, remove wash solution but keep it!

2.b                  Gel slice +

                       500 µl 0.1 M NH4HCO3

                       > 4 h RT, remove wash solution but keep it!

3.                    Gel slice +

                       150 µl 0.1 M NH4HCO3

4.                    + 5 µl 45 mM dithiothreitol

5.                    20 min 50°C, cool

6.                    + 5 µl 100 mM iodoacetamide

7.                    20 min RT

8.                    + 60 µl water

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

10.                  24 h 37°C

11.                  + 500 µl 0.1 M NH4HCO3

                       to extract the peptides, 8h RT,

12.                  + 300 µl 2M urea, 0.1 M NH4HCO3

                       to extract the peptides, 24 h, RT,

13.                  Pool solution from extractions and dry in Speed-Vac

14.                  + 210 µl water, filter through 0.22 µm filter