Mini protean tgx 4 15 tris glycine gels

Protein samples were resolved by SDS-PAGE using Mini-Protean TGX 4–15% Tris-Glycine gels (BioRad) and transferred to a polyvinylidene difluoride (PVDF) membrane (BioRad). After transfer, the PVDF membrane was blocked with 3% BSA in TBST for 1 hour at room temperature. The membrane was washed with TBST (3X) and immunoblotting was performed with the following primary and secondary antibodies at the indicated dilutions: PTP-S-DMD AB (1:200 – 1:1000), SHP2 (Santa Cruz Biotechnology; 1:200), goat anti-rabbit IgG-HRP (Calbiochem, 1:1000 – 1:50000), and rabbit anti-mouse IgG-HRP (Invitrogen, 1:35000) The PVDF membranes were washed with TBST (3X) and developed with ECL Plus chemiluminescence (Pierce) and imaged by film. Equal loading of recombinant phosphatases were assessed by treating the membrane with a solution of 1:1 MeOH:R-250 Coomassie blue for ten minutes, then allowing membrane to dry.

Protein samples were resolved by SDS-PAGE using Mini-Protean TGX 4–15% Tris-Glycine gels (BioRad) and transferred to a polyvinylidene difluoride (PVDF) membrane (BioRad). After transfer, the PVDF membrane was blocked with 5% BSA in TBST for 1 hour at room temperature. The membrane was washed with TBST (3X) and immunoblotting was performed with the following primary and secondary antibodies at the indicated dilutions: HRP-streptavidin (GE Healthcare, 1:80000), Actin (Santa Cruz Biotechnology, 1:1000), PARK7/DJ-1 (Abcam, 1:1000), oxidized PARK7/DJ-1 (Abcam, 1:1000), rabbit anti-goat IgG-HRP (Invitrogen, 1:2000 – 1:50000), and rabbit anti-mouse IgG-HRP (Invitrogen, 1:20000 – 1:50000). The PVDF membranes were washed with TBST (3X) and developed with ECL Plus chemiluminescence (Pierce) and imaged by film.