Reduction and alkylation (Method)
1- Add 1/100 vol. of 0.5M DTT (Dithiothreitol) (77mg/ml) to the sample buffer (e.g. 10ml DTT to 1ml buffer).
2- Dissolve sample in cracking buffer or add 1 volume of cracking buffer to 1 volume of sample.
3- Boil 3min.
4- Apply sample to gel or alkylate by incubating 1min at 100C with 1/20 vol. (based on cracking buffer) of 0.25M iodoacetamide; then apply sample to gel.
Method from: Lane, L.C. A simple method for stabilizing Protein-Sulfhydryl Groups during SDS- Gel Electrophoresis Analytical Biochemistry 86, 655-664 (1978)
Comments
Sample buffers of pH 8-9 are preferable to pH 6.8 Laemmli sample buffer
DTT stock solutions rapidly oxidize - 10s of hours to days (even when frozen)
Be sure to add excess iodoacetamide
See example