Protein Protocols Protein Protocols

532 Fernandez and Mische
membrane appears to be quantitative, and the greatest loss of sample tends to be in the
electroblotting.
10. The entire procedure can be done in approx 24 h plus the time required for peptide mapping
by reverse-phase HPLC. Cutting the membrane takes about 10 minutes, reduction
with DTT takes 30 min, carboxyamidomethylation take another 30 min, digestion at 37°C
takes 22–24 h, and extraction of the peptides requires about 20 min.
References
1. Aebersold, R. H., Leavitt, J., Saavedra, R. A., Hood, L. E., and Kent, S. B. (1987) Internal
amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis
after in situ protease digestion on nitrocellulose. Proc. Natl. Acad. Sci. USA 84,
6970–6974.
2. Tempst, P., Link, A. J., Riviere, L. R., Fleming, M., and Elicone, C., (1990) Internal
sequence analysis of proteins separated on polyacrylamide gels at the submicrogram level:
improved methods, applications and gene cloning strategies. Electrophoresis 11, 537–553.
3. Bauw, G., Van Damme, J., Puype, M., Vandekerckhove, J., Gesser, B., Ratz, G. P.,
Lauridsen, J. B., and Celis, J. E. (1989) Protein-electroblotting and -microsequencing strategies
in generating protein data bases from two-dimensional gels. Proc. Natl. Acad. Sci.
USA 86, 7701–7705.
4. Aebersold, R. (1993) Internal amino acid sequence analysis of proteins after in situ protease
digestion on nitrocellulose, in A Practical Guide to Protein and Peptide Purification
for Microsequencing, 2nd ed. (Matsudaira, P., ed.), Academic, New York, pp. 105–154.
5. Fernandez, J., DeMott, M., Atherton, D., and Mische, S. M. (1992) Internal protein
sequence analysis: enzymatic digestion for less than 10 µg of protein bound to polyvinylidene
difluoride or nitrocellulose membranes. Analyt. Biochem. 201, 255–264.
6. Fernandez, J., Andrews, L., and Mische, S. M. (1994) An improved procedure for
enzymatic digestion of polyvinylidene difluoride-bound proteins for internal sequence
analysis. Analyt. Biochem. 218, 112–117.
7. Fernandez, J., Andrews, L., and Mische, S. M. (1994) A one-step enzymatic digestion
procedure for PVDF-bound proteins that does not require PVP-40, in Techniques in Protein
Chemistry V (Crabb, J., ed.), Academic Press, San Diego, pp. 215–222.
8. Best, S., Reim, D. F., Mozdzanowski, J., and Speicher, D. W., (1994) High sensitivity
sequence analysis using in-situ proteolysis on high retention PVDF membranes and a
biphasic reaction column sequencer, in Techniques in Protein Chemistry V (Crabb, J., ed.),
Academic, New York, pp. 205–213.
9. Atherton, D., Fernandez, J., and Mische, S. M. (1993) Identification of cysteine residues at
the 10 pmol level by carboxamidomethylation of protein bound to sequencer membrane
supports. Analyt. Biochem. 212, 98–105.
10. Kirchner, M., Fernandez, J., Agashakey, A., Gharahdaghi, F., and Mische, S. M. (1996) in
Techniques in Protein Chemistry VII (Marshak, O., ed.), Academic, New York (in press).
11. Atherton, D., (1989) Successful PTC amino acid analysis at the picomole level, in Techniques
in Protein Chemistry (Hugli, T., ed.) Academic, New York, pp. 273–283.
12. Mozdzanowski, J. and Speicher, D. W. (1990) Quantitative electrotransfer of proteins from
polyacrylamide gels onto PVDF membranes, in Current Research in Protein Chemistry:
Techniques, Structure, and Function (Villafranca, J., ed.), Academic, New York, pp. 87–94.
13. Tiller, G. E., Mueller, T. J., Dockter, M. E., and Struve, W. G. (1984) Hydrogenation of
Triton X-100 Eliminates Its Fluorescence and Ultraviolet Light Absorbance while Preserving
Its Detergent Properties. Analyt. Biochem. 141, 262–266.