assay protocol

Conformational Stability Assay

This conformational stability assay is based on the denaturation affect of Gdn-HCl on proteins. Gdn-HCl is one of the strongest denaturants used in biochemical studies of protein folding. In 6M Gdn-HCl solution, all proteins become randomly coiled.

Procedure for conformational stability assay:

1. Mix 100μl of the purified proteins with five volumes of cold methanol at -20°C for 2h.

2. After centrifuged at 20,000g for 30 min,

3. Resuspended the pellets with 100 μl of different concentration of GdnHCl, e.g. 1, 2, 3, 4, 5 and 6M and incubated at 37°C for 12 h.

4. Add five volumes of cold methanol into each preparation and maintained at -20°C for 2h.

5. Centrifuged at 20,000g for 30 min, and resuspended the pellets with 100μl TN buffer (10 mM Tris, 130 mM NaCl, pH 7.0).

6. A half is employed directly into SDS-PAGE and the rest is subjected into PK-digestion (50 μg/ml) at 37°C for 1h.

Proteinase K digestion:

Incubate your proteins with suitable and gradient concentrations of proteinase K at 37°C for 1h, then proceed to a western-blot test or a SDS-PAGE (followed with a Coomassie-blue staining) to valuate the protein fragments left.