Protein Protocols Protein Protocols

676 Dowdy
3.3. Trypsinization of Protein on Nitrocellulose Filter
1. Following transfer of 32 P-labeled proteins to NC filter, open the transfer unit. Using a pair
of filter forceps, place the NC filter, protein side down onto Saran wrap, and cover. Expose
the Saran wrap-covered NC filter to ARG film for approx 1–2 h with protein side toward
the film. The length of the exposure will vary with the abundance of 32 P-content and
protein, and with the efficiencies of recovery from immunoprecipitation and transfer.
Radioactive or luminescent markers are needed on the filter to determine the orientation,
position, and alignment of the NC filter with respect to the ARG film. Using the markers,
line up your filter on top of its ARG. This is best done on a light box. Use a razor blade to
excise the slice of NC filter that corresponds to the 32 P-labeled protein band.
2. Place the NC filter slice into an Eppendorf tube, add ~200 µL of 0.5% PVP-360 in 100 mM
acetic acid, cap tube, and incubate in a shaking water bath at 37°C for 30 min (see Note 3).
3. Wash the filter slice five times with 1 mL H 2O and then twice with 1 mL fresh 50 mM
NH 4HCO 3.
4. Add 10 µg of trypsin in 150 µL of 50 mM NH 4HCO 3 to the NC filter slice. Incubate in a
37°C shaking water bath overnight. Following this incubation, spike the digestion of the
NC filter with another 10 µg of trypsin in 150 µL of 50 mM NH 4HCO 3, and incubate at
37°C for an additional 4 h.
5. Vortex the tube containing the NC filter/trypsin sample for 1 min, and centrifuge at 12,000
and 4°C for 30 s. Transfer the supernatant to a new Eppendorf tube. Wash the NC filter
slice by addition of 300 µL H 2O, vortex for 1 min, centrifuge at 12,000 for 30 s, and
combine the supernatants.
6. Freeze the trypsinized 32 P-labeled peptide sample on dry ice and then completely dry in a
Speed Vac (without heat). This generally takes about 4 h to complete. Prepare the
performic acid for the oxidation step (step 8).
7. After the sample has been completely dried, add 50 µL of ice-cold performic acid, and
place on ice for 1 h. Stop the reaction by addition of 400 µL H 2O to the sample, followed
by freezing on dry ice and then drying in a Speed Vac.
8. Resuspend the sample in 8–10 µL of H 2O. Determine the level of radioactivity by counting
0.5 µL of the sample on a scintillation counter. Usually a total of ≥2000 cpm is sufficient
for 2-D phosphopeptide mapping.
3.4. Phosphopeptide Separation: First and Second Dimensions
1. Mark the origin on the TLC plate by lightly touching a pencil to the TLC plate at the
position indicated in Fig. 1. Apply multiple 0.5-µL aliquots of the trypsinized 32 P-labeled
peptides to the origin to achieve ≥2000 cpm. Dry the TLC plate thoroughly between each
aliquot application by use of a gentle fan. Pay particular attention to adding each subsequent
aliquot to the same small area at the origin. Add 0.5 µL of the color marker 3 cm
from the top edge of the TLC plate (Fig. 1) and then dry the TLC plate under a fan for an
additional 30–60 min.
2. Prepare the Multiphor II apparatus for electrophoresis. Place the Multiphor II in a cold
room, connect the cooling plate to the cooling circulator bath hoses, and precool to 5°C.
Prepare and chill the pH 1.9 running buffer, and add to Multiphor II buffer tanks. Insert
the electrode paddles into innermost chambers, and attach the wire connections. We have
used the IEF electrodes in direct contact with the cellulose plate; however, using the
paddles provided and wicking buffer onto the plate yield the best results (see Figs. 1 and 2).
Place the cooling plate into the Multiphor apparatus. Add 1 L of prechilled pH 1.9 running
buffer to each chamber of the Multiphor II. (These instructions are provided with the
Multiphor II unit.)