This procedure was adapted from the following article:
Wong, S. C., Grimley, C., Padua, A., Bourell, J. H., and Henzel,
W. J. (1993) Peptide Mapping of 2-D Gel Proteins by Capillary HPLC. Tech. In
Prot. Chem. IV, 371-378
Reagents:
- Methanol
- Acetonitrile
- Water
- DTT, Clealands reagent
- Tris-Base
- HCl
- Promega modified trypsin
- Iodoacetic acid (IAA) (see
tips on this reagent, step 4 on this link)
- PVP, polyvinylpyrolodone
Solutions and Stocks:
- 1 M DTT (in water, keep this stock frozen at -20 °C)
- 0.5 M Tris-Base / 6M guanidine-HCl / 5 mM EDTA / 10% acetonitrile / 7 mM
DTT / pH 8.5 with HCL (note: pH with HCl before adding
the acetonitrile, starts out around pH 10. Add DTT fresh to a portion of
this solution right before reduction)
- 200 mM IAA in 0.5 M NaOH make fresh each time
- 10% Acetonitrile
- 0.25% PVP in 0.1% acetic acid
- 100 mM Tris-Base, pH 8.0, 10 % acetonitrile
Procedure:
- Cut out the band or spot as narrowly as possible, avoid touching the blot,
wear gloves.
- Wet excised blot with 1-3 µL of methanol in a small
tube. (the membrane is very hydrophobic and must
be wetted with methanol. Do not let the membrane dry out during the
procedure.)
- Reduction. Reduce with 100 µL of Solution 2 at 45
°C for 60 min.
- Cool and add 10 µL of Solution 3, let react for 20 min. in
the dark.
- Wash the blot 3X with 500 µL Solution 4.
- To block the blot incubate in 200 µL of Solution 5 for 20
minutes on a room temp. shaker.
- Wash the blot 3X with 500 µL Solution 4.
- Transfer blot to new tube.
- Wash the blot 3X with 500 µL Solution 4. (this
extensive wash and transfer is necessary to remove excess PVP 360, which
will give UV and mass spectral interference if not removed.)
- Digest with 0.2 µg Promega modified trypsin in 100 µL of
Solution 6 for 24 hours at 37 °C.
- Concentrate on a SpeedVac to 20 µl, inject 5 µL onto a
capillary column for LC/MS analysis.
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Figure 1 |
