ATTENTION: All solutions need to be made FRESH right before the digestion. High purity reagents preferred (99+%).

Eppendorf tubes must be siliconized.  One suggestion: Polypropylene Eppendorf Tube, Sarstadt 72.690 & 72.699, Tel: 1-800-257-5101.

50% ACN in dd H₂O

250 µl Acetonitrile + 250 µl doubly deionized H₂O

100 mM Ambic

100 µl of 1M Ammonium Bicarbonate (0.79 mg in 10 ml dd H₂O) in 900 µl of H₂O

25 mM Ambic

25 µl 1M Ambic + 975 µl dd H₂O

10 mM DTT in 100 mM Ambic

5 µl 2M Dithiothreitol (0.15 mg in 500 µl dd H₂O) + 895 µl dd H₂O + 100 µl 1M Ambic

55 mM IAA in 100 mM Ambic

10 mg Iodoacetamide + 100 µ l 1M Ambic + 900 µl dd H₂O

Trypsin solution

5 µl 1M CaCl₂ + 50 µl 1M Ambic + 945 µl dd H₂O + 125 µl 0.1 µg/µl trypsin in dd H₂O 
Must be sequencing grade trypsin.  One suggestion:  B-Mannheim sequencing grade, Modified Trypsin (highly purified), pH 7.5~9,  Catalogue 1418033

Buffer solution without trypsin

5 µl 1M CaCl ₂ + 50 µl 1M Ambic + 945 µl dd H₂O 

10 mM DTT in dd H ₂O

5 µl 2M DTT + 995 µl dd H₂O

5% Formic Acid

57 µl stock Formic Acid (~88%) + 943 µl dd H₂O

Stock Solutions

1M Ambic

0.79 g Ammonium Bicarbonate in 10 ml dd H₂O

2M DTT 

0.15 g DTT in 500 µl dd H₂O

1M CaCl₂

0.15 g CaCl₂·2H₂O in 1ml dd H₂O

30 mM Potassium Ferricyanide

10 mg Potassium Ferricyanide in 1 ml dd H₂O

100 mM Sodium Thiosulfate

25 mg Na₂S₂O₃·5H₂O in 1 ml dd H₂O