SDS-PAGE analysis of the denatured soluble CT domain with (+) and without (-) the reducing agent dithiothreitol (DTT).


METHODS
For CT in soluble form, the Laemmli SDS sample buffer (Bio-Rad), with or without the reducing agent dithiothreitol (DTT, final concentration of 10 mM), was combined with the sample and left for incubation at 95 oC for 10 min before protein electrophoresis. SDS-PAGE was carried out using Mini-PROTEAN® TGX™ precast gels 4–20% (Bio-Rad), 1x tris-glycine-SDS running buffer for 36 min, at 180 volts and 4 °C. As reference, the PageRuler (Thermo Scientific PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa) was used. SimplyBlue SafeStain (Thermo Scientific) was used to stain the gels.

For analysis of the CT domain in fiber form, the fiber was first dissolved with 6 M urea. Laemmli SDS sample buffer (Bio-Rad) without reducing agent was added and left for incubation at 95 oC for 10 min before protein electrophoresis using Mini-PROTEAN® TGX™ precast gels 4–20% (Bio-Rad), 1x tris-glycine-SDS running buffer for 36 min, at 180 volts. Here, Bio-Rad Precision Plus Protein™ All Blue Prestained Protein Standard was used as ladder. Bio-Safe™ Coomassie Stain was used to stain the gel.


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