Syprored protein gel stain

SDS-PAGE gels run for this study were done using a Tris-Tricine buffer system (200 mM Tris, 100 mM Tricine, 0.1% SDS, pH 8.3) to better resolve low molecular weight proteins (<20 kDa) (57 (link)). Protein visualization on SDS-PAGE gels was done with the SYPRO Red protein gel stain (Invitrogen). The gel was rinsed briefly in deionized water before being stained for 1 h with 1:5,000 SYPRO Red (Invitrogen) diluted in 10% (vol/vol) acetic acid. The gel was then destained for 15 min in 7.5% (vol/vol) acetic acid before being imaged on a Chemidoc imaging system (Bio-Rad). For Western blots, the resolved proteins were transferred to a nitrocellulose membrane by wet transfer (100 V, 30 min). Nitrocellulose membranes were then blocked with 5% skim milk dissolved in TBS-T for 30 min with light agitation followed by addition of primary antibody (titer 1:5,000) to the blocking buffer and further incubation for 1 h. Blots were washed for 5 min three times with TBS-T then incubated in TBS-T with an HRP-conjugated antirabbit secondary antibody (titer 1:5,000) for 45 min. After three additional 5-min washes, the blots were developed using Clarity Max Western ECL reagent (Bio-Rad) and imaged with a ChemiDoc XRS+ (Bio-Rad).