Protein Protocols Protein Protocols

Enzymatic Digestion of Membrane-Bound Protein 527
2. 100 mM iodoacetamide solution: bring 9.25 mg iodoacetamide (reagent-grade) up in
500 µL HPLC-grade water. This solution must be made fresh just prior to use. Dry DTT
and iodoacetamide should be stored at 4°C or –20°C.
2.4. Enzyme Solutions and Inhibitors
Enzymes should be stored as small aliquots at –20°C, and made up as 0.1 µg/µL
solutions immediately before use. These aliquots can be stored for at least 1 mo at –20°C
without significant loss of enzymatic activity.
1. Trypsin (25 µg, sequencing-grade, Boehringer Mannheim, Indianapolis, IN): Solubilize
trypsin in 25 µL of 0.01% trifluoroacetic acid (TFA), and let stand ~10 min. Aliquot 5 µL
(5 µg) quantities to clean microcentrifuge tubes, dry in a SpeedVac, and store at –20°C.
Reconstitute the dry enzyme in 50 µL 0.01% TFA for a 0.1 µg/µL working solution, which
is good for 1 d. Trypsin cleaves at arginine and lysine residues.
2. Endoproteinase Lys-C (3.57 mg, Wako Pure Chemicals, Osaka, Japan): Solubilize enzyme
in 1000 µL HPLC-grade water, and let stand ~10 min. Make nine 100-µL quantities to clean
tubes, and store at –20°C. Disperse remaining 100 µL into 20 × 5 µL aliquots (17.85 µg each),
and store at –20°C. When needed, take one 5-µL aliquot and add 173 µL of HPLC-grade
water to establish a 0.1 µg/µL working solution, which can be used for up to 1 wk if stored
at –20°C between uses. When 5-µL aliquots are used up, disperse another 100-µL aliquot.
Endoptoteinase Lys-C cleaves only at lysine residues.
3. Endoproteinase Glu-C (50 µg, sequencing-grade, Boehringer Mannheim) : Solubilize in
100 µL of HPLC-grade water, and let stand ~10 min. Aliquot 10-µL (5 µg) quantities to
clean tubes, dry in a SpeedVac, and store at –20°C. Reconstitute the enzyme in 50 µL HPLCgrade
water for a 0.1 µg/µL solution, which is good for only 1 d. Under these conditions,
endoproteinase Glu-C cleaves predominantly at glutamic acid residues, but can sometimes
cleave at aspartic acid residues.
4. Clostripain (20 µg, sequencing-grade, Promega, Madison, WI): Solubilize enzyme in
200 µL of manufacturer’s supplied buffer for a concentration of 0.1 µg/µL. Enzyme
solution can be stored at –20°C for 1 mo. Clostripain cleaves at arginine residues only.
5. 1% Diisopropyl fluorophosphate (DFP) solution: DFP is a dangerous neurotoxin that must
be handled with double gloves in a chemical hood. Please follow all precautions listed
with this chemical. Add 10 µL of DFP to 990 µL absolute ethanol in a capped microcentrifuge
tube. Store at –20°C.
3. Method
3.1. Preparation of the Membrane-Bound Sample
Protein should be electrophoresed in one or two dimensions, followed by electrophoretic
transfer to the membrane and visualization of the bands according to the following
suggestions.
1. Electroblotting of proteins will be most efficient with a tank transfer system, rather than a
semidry system. Concentrate as much protein into a lane as possible; however, if protein
resolution is a concern, up to 5 cm 2 of membrane can be combined for digestion (see
Notes 3 and 5).
2. Stain PVDF membrane with either Ponceau S, Amido black, india ink, or chromatographically
pure Coomassie brilliant blue with a dye content of 90%. Destain the blot until
the background is clean enough to visualize the stained protein. Complete destaining of
the blot is unnecessary, but at least three washes (~5 min) with distilled water should be
done to remove excess acetic acid, which is used during destaining (see Note 5).