Irdye 680lt goat anti rabbit igg secondary antibody

Proteins from cells expressing low or high levels of mCherry-SKL were extracted by Urea protein extraction. In short, 10 mL of 0.5 OD₆₀₀ yeast cells were harvested by centrifugation. Next, 200 µL of lysis buffer containing 8 M Urea, 50 mM Tris, protease inhibitor (Merck) pH = 7 were added to the pellet and the cell wall was broken down by vortexing at high speed with ready-to-use glass beads (Scientific Industries) at 4 °C for 10 min; 25 µL of 20% SDS were added and the extracts were incubated at 95 °C for 5 min. Protein extracts were transferred into new tubes and stored at −20 °C. Samples in Fig. 1D were analyzed by SDS/PAGE and Western blotting using an anti-Pex5 antibody 1:10,000 (44 (link)) (kindly provided by Ralf Erdmann, Ruhr-Universität Bochum, Bochum, Germany), and anti-Histone H3 (Abcam, # ab1791; 1:10,000) was used as the loading control. The anti-Histone H3 antibody was diluted in a mix of 3% BSA, 0.01% sodium azide and Phenol red in PBS (Biological Industries) solution. IRDye 680LT goat anti-rabbit IgG secondary antibody (LI-COR Biosciences) was used at 1:10,000 dilution, followed by scanning using the Odyssey Imaging System (LI-COR Biosciences).