Anti mouse hrp conjugate

Prior to MS, 1 µl of streptavidin bead eluent was spotted onto a methanol-activated polyvinylidene difluoride (PVDF) membrane and allowed to dry completely. The membrane was then reactivated in methanol, washed in water, and post-fixed in 4% PFA for 30 min. Blots were then rinsed with TBST (20 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween-20) and placed in blocking buffer (TBST and 5% BSA) for 1 h at room temperature. To detect biotinylated proteins, blots were incubated with ABC (VectorLabs) diluted 1:10 in a blocking buffer for 1 h at room temperature. To detect alpha-synuclein, blots were incubated overnight with SYN1 diluted 1:2000 in blocking buffer, washed, and then incubated with anti-mouse HRP conjugate diluted 1:6000 in blocking buffer (Cell Signaling). All membranes were washed in high stringency wash buffer (20 mM Tris-HCl pH 7.6, 400 mM NaCl, 0.1% Tween-20) and imaged using enhanced chemiluminescence (ECL) substrate (Bio-Rad, product # 1705060) and Chemidoc imager (Bio-Rad).

15–20 μg of protein were separated using 4–13% Bis-tris gels, blotted onto methanol-activated polyvinylidene difluoride (PVDF) membrane, post-fixed with 4% PFA for 30 min, and then allowed to dry completely. Blots were then reactivated in methanol and stained for total protein (Licor). For CIAP treatment, blots were blocked in 2% (w/v) polyvinylpyrrolidone (PVP) for 1h, and then incubated with CIAP as described above. Blots were then rinsed with TBST (20 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween-20) and placed in blocking buffer (TBST and 5% BSA or 5% dry milk) for 1 h at room temperature. Blots were incubated overnight with PSER129 diluted 1:50,000 or SYN1 diluted 1:2000 in blocking buffer. Blots were then washed and incubated with anti-rabbit HRP conjugate (dil. 1:20,000, Invitrogen), anti-mouse HRP conjugate (dil. 1:6,000, Cell signaling), or anti-mouse IRDye 680RD (dil. 1:20,000 Li-Cor) diluted in blocking buffer for 1 h. Membranes were washed again in TBST and imaged using enhanced chemiluminescence (ECL) substrates (Bio-Rad, product # 170-5060 or ThermoFisher Scientific, product #38554) with a Chemidoc imager (Bio-Rad) or odyssey M imager for fluorescence (Li-Cor).