Anti β tubulin monoclonal antibody

The proteins in an SDS-polyacrylamide gel were transferred onto an Immobilon^®-P PVDF transfer membrane (Millipore Sigma) by electroblotting at 250mA for two hours in ice cold transfer buffer (25mM Tris, 192mM glycine and 10% methanol). The blots were blocked at room temperature for one hour in Blotto (PBT: 10% PBS and 0.1% Tween-20, and 3% skim milk). Anti-FLAG M2 monoclonal antibody (Sigma-Aldrich) at a dilution of 60,000-fold in Blotto was incubated with the blot for one hour at room temperature. After washes with PBT, horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (ThermoFisher Sci.) at a dilution of 3,000-fold in Blotto was added and incubated for one hour at room temperature. After washes with PBT, HRP was detected using SuperSignal^™ West Femto Maximum Sensitivity Chemiluminescent Substrate (ThermoFisher Sci.). Digital images were recorded using a ChemiDoc^™ Imaging System (Bio-Rad). For some experiments, the membrane was stripped for one hour at room temperature using Restore^™ Western Blot Stripping Buffer (ThermoFisher Sci.) to remove the anti-FLAG antibody and was blocked at room temperature for one hour in Blotto followed by incubation with anti-β-tubulin monoclonal antibody (E7 concentrated from Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) at a dilution of 1,500-fold in PBT.

The primary antibodies used in this study were anti-Flag M2 monoclonal antibody (1:5,000 dilution for western blotting and 1:1,000 dilution for immunofluorescence; Sigma), anti-Myc monoclonal antibody (1:5,000 dilution, 9E10; Developmental Studies Hybridoma Bank), anti-BmVasa antibody [1:20 dilution (supernatant), 1C3D10^{11 (link)}], anti-Ago3 antibody (1:500 dilution for immunofluorescence, 7A7^{11 (link)}), and anti-β-Tubulin monoclonal antibody (1:1,000 dilution, E7; Developmental Studies Hybridoma Bank). The secondary antibodies used in this study were Peroxidase-conjugated anti-mouse IgG (1:5,000 dilution; Cappel), TrueBlot ULTRA: Anti-Ig HRP, Mouse (Rat), eB144 (1:1,000 dilution; ROCKLAND), Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (1:1,000 dilution; Invitrogen), and Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555 (1:1,000 dilution; Invitrogen).