Protein Protocols Protein Protocols

Cysteine Residues and Disulfide Bonds 601
3.3. Electrophoretic Analysis
1. 50 µL aliquots of each sample, Labeled 1–6, mixed with 12 µL of appropriate tracking dye
solution, are loaded onto successive lanes of a polymerized high- or low-pH gel, set up in
a suitable slab gel electrophoresis apparatus.
2. Low-pH buffer system: Electrophoresis is carried out toward the negative electrode, using
a current of 5–20 mA for each gel, overnight at 8°C.
3. High-pH buffer system (10); Electrophoresis is carried out toward the positive electrode
at 10–20 mA per gel (or 100–180 V) for 3–4 h.
4. Electrophoresis is stopped when the tracking dye reaches bottom of the gel.
5. Proteins are visualized using conventional stains e.g., Coomassie blue (Pierce, Chester,
UK), silver staining (see Chapters 33).
4. Notes
1. The method of Takahashi and Hirose (6) can be used to categorize the half-cystines in a
native protein as:
a. Disulfide bonded;
b. Reactive sulfhydryls; and
c. Nonreactive sulfhydryls.
In the first step, the protein sulfhydryls are alkylated with iodoacetic acid in the presence and
absence of 8 M urea. In the second step, the disulfide bonded sulfhydryls are fully reduced
and reacted with iodoacetamide. The method described above is then used to give a ladder
of half-cystines so that the number of introduced carboxymethyl groups can be quantified.
2. Urea is an unstable compound; it degrades to give cyanates which may react with protein
amino and thiol groups. For this reason, the highest grade of urea should always be used,
and solutions should be prepared immediately before use.
3. Electrophoresis in gels containing higher or lower percent acrylamide may have to be
employed depending on the molecular weight of the particular protein being studied.
4. Where protein is already in solution, it is important to note that the pH should be adjusted
to around 8.0, and the DTT and urea concentrations should be made at least 10 mM and
8 M, respectively.
5. Other ratios of iodoacetic acid to iodoacetamide may need to be used if more than about
eight cysteine residues are expected, since a sufficiently intense band corresponding
to every component in the complete range of charged species may not be visible. A
greater ratio of iodoacetic acid should be used if the more acidic species are too faint (and
vice versa).
References
1. Creighton, T. E. (1980) Counting integral numbers of amino acid residues per polypeptide
chain. Nature 284, 487,488.
2. Freedman R. B., Hirst, T. R., and Tuite, M. F. (1994) Protein disulphide isomerase: building
bridges in protein folding. Trends Biochem. Sci. 19, 331–336.
3. Creighton, T. E. (1989) Disulphide bonds between cysteine residues, in Protein
Structure—a Practical Approach (Creighton, T. E., ed.), IRL, Oxford, pp. 155–167.
4. Freedman, R. B. (1984) Native disulphide bond formation in protein biosynthesis; evidence
for the role of protein disulphide isomerase. Trends Biochem. Sci. 9, 438–441.
5. Feinstein, A. (1966) Use of charged thiol reagents in interpreting the electrophoretic
patterns of immune globulin chains and fragments. Nature 210, 135–137.