Western Blotting of Trypanosome Proteins

Western botting was performed according standard protocols, using ∼2 × 10⁶ parasites. TbPolIE-12myc was detected using mouse anti-myc clone 4A6 antibody at 1:7000 (Millipore). The detection of Ef1α (loading control), was performed with the mouse anti-Ef1α clone CBP-KK1 antibody at 1:25 000 (Millipore). Both antibodies were used in combination with the goat anti-mouse IgG (H+L) horseradish peroxidase conjugate at 1:3000 (Life Technologies). While the damage accumulation marker γH2A (42 (link)) was detected using rabbit anti-γH2A at 1:10 000 in combination with goat anti-rabbit IgG (H+L) horseradish peroxidase conjugate at 1:3000 (Life Technologies). To analyse VSG expression, cell lysates of ∼2 × 10⁶ cells were separated by SDS-PAGE and transferred to a PVDF membrane. VSGs were detected using polyclonal antibodies from rabbits immunized with VSG2, VSG3, VSG6 or VSG13 (gift from Markus Engstler, University Wuerzburg). Signal intensity was normalized using a monoclonal antibody L13D6 specific for a paraflagellar rod protein (Gift from Keith Gull, University of Oxford). IRDye680- or IRDye800-conjugated secondary antibodies were used in combination with an Odyssey infrared scanner (LI-COR).