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).
Goat anti mouse igg h l horseradish peroxidase conjugate
Manufactured by Thermo Fisher Scientific
Goat anti-mouse IgG (H+L) horseradish peroxidase conjugate is a secondary antibody used in various immunoassays and immunochemical techniques. It binds to mouse primary antibodies and is conjugated with the enzyme horseradish peroxidase, which can be used for signal detection.
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2 protocols using goat anti mouse igg h l horseradish peroxidase conjugate
1
Western Blotting of Trypanosome Proteins
2
Immunodetection of Tagged Proteins
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Approximately 2.5 × 106 PCF or BSF cells were collected for immunodetection of the proteins of interest by western blot. The 12myc-tagged proteins were detected using the mouse anti-myc clone 4A6 antiserum (diluted 1:7000; Millipore), while 6HA-tagged proteins were detected using the mouse anti-HA clone HA-7 antiserum (diluted 1:10 000; Sigma-Aldrich). Whenever necessary, the transcription elongation factor Ef1α was used as a loading control and detected with the mouse anti-Ef1α clone CBP-KK1 antiserum (diluted 1:25 000; Millipore). The three antisera were used in combination with the goat anti-mouse IgG (H+L) horseradish peroxidase conjugate (diluted 1:5000; Life Technologies).
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