Peroxidase affinipure goat anti rabbit mouse igg

Cell protein extracts were obtained by means of RIPA lysis buffer. Equal amounts of solubilized proteins were heated in Laemmli sample buffer (Bio‐Rad) containing 2‐βmercaptoethanol (70 mM, Sigma), separated by SDS‐PAGE gel 10% and electroblotted onto a nitrocellulose membrane (GE Healthcare, Amersham™). Membranes were fixed for 30 min in 0.4% paraformaldehyde (Lee & Kamitani, 2011 (link)). Membranes were blocked in 5% slim milk (diluted in TBS with 0.05% Tween‐20) and probed with the appropriate primary antibody against α‐syn (AB138501; abcam) and GAPDH (2118; Cell Signaling) overnight at 4°C. The membrane was then incubated with specific secondary antibody Peroxidase AffiniPure Goat Anti‐Rabbit/Mouse IgG (Jackson). Proteins were visualized by means of an enhanced chemiluminescence detection system (ECL™; Amersham). After acquisition by a GelDoc™ image capture system (Kodak), proteins were quantified using ImageJ software.

Samples were homogenized and extracted in RIPA lysis buffer with a phosphatase and protease inhibitor cocktail, following 10 min boiling and centrifugation to collect the supernatant for subsequent analyses. The protein concentration was determined using a BCA Protein Quantification Kit. Samples containing equal amounts of protein were loaded and separated on 10% SDS-PAGE and subsequently transferred to polyvinylidene difluoride membranes. Membranes were incubated with primary antibodies overnight at 4 °C. After washing three times with TBST, the membranes were incubated with Peroxidase-AffiniPure Goat Anti-Rabbit/Mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) at a dilution of 1:1000 for 1 h. Then, the membranes were washed another three times with TBST. Finally, the blots were visualized using an X-ray film processor (Delight, Suzhou, China). Protein band intensities were quantified in Image J (version 1.6, NIH, Bethesda, MD, USA) software.