Protein Protocols Protein Protocols

Enzymatic Digestion 515
Fig. 1. In-gel lysyl endopeptidase digestion of transferrin. Following SDS-PAGE of 25 pmol
of transferrin in a 12.5% polyacrylamide gel, the gel was stained and destained as described in
Subheading 3. The protein band was then excised, along with a control blank, and digested in
the gel with lysyl endopeptidase as described in Subheading 3. Peptides were chromatographed
on a Vydac C-18, 2.1 × 250 mm reverse-phase column that was eluted at a flow rate of 150 µL/min
as described in Chapter 72. A comparison of the transferrin digest (top profile) with the control
digest (bottom profile), which was carried out on a blank section of gel, indicates that the digest
proceeded well.
Although the following procedure, which is a modification of the Rosenfeld et al.
(4) procedure as reported in Williams and Stone (3), is capable of succeeding with as
little as 25 pmol protein (e.g., see Fig. 1), the minimum amount of protein recommended
for in gel digestion is 50–100 pmol at a minimum protein to gel density (after
staining/destaining) of 0.05 µg/mm 3 (see Notes 7 and 9). The protein of interest, along
with a blank section of gel that serves as a control, are excised using a razor blade and
tweezers. To prevent going ahead with insufficient protein, we recommend that a
10–15% section of the gel band of interest be subjected to hydrolysis/ion exchange
amino acid analysis to quantitate the amount of protein remaining (see Note 10).
3.1.2.2. IN-GEL DIGESTION OF PROTEINS WITH TRYPSIN AND LYSYL ENDOPEPTIDASE
1. Determine the approximate volume of gel to be digested (length × width × thickness).
2. Cut the gel band(s) containing the protein of interest into approx 1 × 2 mm pieces, and
place in an Eppendorf tube. Repeat for the “blank” section of gel.
3. Add 150 µL (or more, if necessary to cover the gel pieces) 50% CH 3CN/0.1 M Tris-HCl,
pH 8.0, to the gel pieces.
4. Wash for 15 min at room temperature on a rocker table.
5. Remove wash.
6. Semidry the washed gel pieces in a SpeedVac to approx 25–50% of original volume.
7. Make up the enzyme solution by diluting 5 µL 0.1 mg/mL enzyme stock solution with
10 µL 0.1 M Tris-HCl, pH 8.0, 0.1% Tween 20 for every 15 mm 3 gel that is to be digested.
8. Rehydrate gel pieces with the enzyme solution from step 7, which should be equal in volume
to that of the gel pieces and should provide a final enzyme ratio of about 0.5 µg/15-mm 3
gel volume.