Syprorange

Standard Glycine SDS-PAGE was used for the analysis of Plin4 12mer and csw 12mer (Mw ~40 kDa) using homemade 13% acrylamide-bisacrylamide gels. Tricine SDS-PAGE (Schägger and von Jagow, 1987 (link)) was used for proteins with lower molecular weight, i.e. Plin4 4mer, Plin4 4T > S, Plin4 NN, or Plin3 AH (9–15 kDa). For that we either used TruPAGE commercial gels (Sigma) and homemade Tris-MOPS buffer (60 mM Trizma, 30 mM 4-Morpholinepropanesulfonic acid (MOPS), 0.1% w/v SDS), or, for better resolution, homemade 16.5% acrylamide-polyacrylamide (29:1) gels run with tricine buffer (100 mM Tris-HCl pH 8–8.5, 0.1 M Tricine, 0.1% SDS) in the cathode and 200 mM Tris HCl pH 8.9 in the anode chamber. Gels were rinsed in 7.5% acetic acid, stained with SyprOrange (Life Technologies) and visualized with a MP imaging system (Bio-Rad) using the Alexa 488 settings. Because all perilipin AH purified construct lack aromatic residues, preventing protein quantification by UV spectroscopy or by Bradford assay, protein concentration was routinely determined by densitometry of Sypro-Orange or Coomassie Blue stained gels against a calibration curve with protein standards (Sigma) using ImageJ. Quantification by gel electrophoresis was verified by Ellman´s reaction method as previously described (Čopič et al., 2018 (link)).