Anti myc rabbit polyclonal

Antibodies used in this study were: anti-HA mouse monoclonal antibody (Covance Inc., Princeton, NJ, United States), anti-HA rabbit monoclonal (Upstate Cell Signaling Solutions, Waltham, MA, United States), anti-myc rabbit polyclonal, (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, United States), anti-Golgin-97 (Invitrogen, #A21270), anti-p230 (BD Transduction Laboratories, Lexington, KY, United States #611230), anti-GM130 (BD Transduction Laboratories, #51-9001978), horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit (Jackson Laboratory, Bar Harbor, ME, United States), Goat anti-GST-HRP (Amersham Biosciences, Piscataway, NJ, United States), Alexa Fluor-conjugated secondary antibodies (Invitrogen Molecular Probes, Eugene, OR, United States).

Bacterial extracts, trypanosome whole cell protein lysates and purified proteins were separated on SDS-PAGE gels (10%–15%) and transferred by semi-dry (BioRad) blotting 45 min at 25V on PVDF membrane. After a 1 h blocking step in 5% Milk in PBS-0.2% Tween, the membranes were incubated with the primary antibodies diluted in blocking buffer–anti-TbSAXO (mAb25 mouse monoclonal, 1:1,000 dilution [80 (link)]); anti-FPC4 (rat, 1:500 dilution) and anti-myc (rabbit polyclonal (Santa Cruz), 1:500 dilution). After three washes in blocking buffer, the membranes were incubated with the secondary antibodies–anti-mouse antibody HRP-conjugated (Jackson, 1:10,000 dilution), anti-rat antibody HRP-conjugated (Jackson, 1:10,000 dilution) and anti-rabbit antibody HRP-conjugated (Sigma, 1:10,000 dilution)–and washed twice 10 min in blocking buffer and twice 5 min in PBS. Blots were revealed using the Clarity Western ECL Substrate kit (Bio-Rad) with the ImageQuant LAS4000.