Rabbit anti beta tubulin antibody

HeLa cells were lysed two days after transfection in 1x Laemmli buffer with 4 M urea and 50 mM DTT. Proteins were separated by 12% SDS-PAGE and transferred onto Amersham Protran nitrocellulose membrane (0.45 uM, GE Healthcare). Rabbit antiserum raised against MusD1 Gag and recognizing MusD6 Gag proteins was a kind gift from the Heidmann lab (Ribet et al., 2004 (link)). Anti-rabbit HRP-coupled IgG was used as secondary antibody (Jackson Immuno Research) and ECL Western Blotting Substrate (Pierce) for detection. To control for equal loading, membranes were stripped with Restore Stripping Buffer (Pierce) and incubated with rabbit anti-beta Tubulin antibody (Abcam). All antibody incubations were performed in 5% milk in TBS buffer with 0.05% v/v Tween20.

For all the gels shown in this study, proteins separated on NuPAGE 4-12% Bis- Tris gels were transferred to nitrocellulose membrane. Media and plasma samples were further incubated with Ponceau S solution to ensure equal loading and washed with PBST (PBS, 0.05% Tween 20). Blots were blocked in Odyssey blocking buffer for 30 min at room temperature and incubated with primary antibodies (Mouse anti-V5 antibody, Invitrogen R960-25, 1:1000 dilution; Mouse anti-Flag antibody (Sigma F1804), 1:1000 dilution; Rabbit anti-beta Tubulin antibody (Abcam ab6046), 1:5000 dilution; Rabbit anti-beta-Actin (Abcam ab8227), 1:5000 dilution; Rabbit anti-BHMT antibody (Abcam 96415); in blocking buffer for 1 h at room temperature or overnight at 4°C. After washing three times with PBST, blots were stained with species matched secondary antibodies (Streptavidin-AlexaFluor680, ThermoFisher S32358, 1:1000 dilution; Goat anti-mouse IRDye 680RD, LI-COR 925-68070, 1:10000 dilution; Goat anti-rabbit IRDye 800RD, LI-COR 925-68070, 1:10000 dilution. Blots were then washed three times with PBST and imaged with Odyssey CLx Imaging System.