Conformational stability assay and immunoblotting

1. 6M GndHCl made fresh in dH2O
2. GndHCl treatment at 37'C, 1 hr, 800 rpm 
3. Add dH2O to bring down GndHCl concentration to 0.5M
4. PK digestion 1 ug/ml, 37'C, 30 min, 500 rpm 
5. Stop PK digest at 95'C, 10 min 
6. Spin down the samples 100,000 xg, 1 hr, 4 'C 
7. Resuspended the samples in 15 ul of 30 mM Tris-HCl 

SDS-PAGE

1. Tricine sample buffer (BioRad) was added to the samples and boiled at 95’C for 10 min
2. Samples were analysed with 16.5% Tricine gel (BioRad)
3. 180V, 1 hr 

WB

1. SDS-PAGE gels were immunoblotted onto nitrocellulose membranes (Amersham) using wet transfer method
2. Membranes were blocked with 5% skimmed milk in TBS-T
3. Syn1 1:1,000 in 5% BSA overnight at 4’C
4. Anti-mouse 1:5,000 in 5% BSA at RT for 1 hr 
5. Membranes were developed using ECL (Amersham)