Pulse-chase analyses were performed using metabolic radiolabelling with [35S]methionine/cysteine (Perkin Elmer, Waltham, MA, USA) and subsequent immunoprecipitation of specific radiolabeled proteins were conducted as previously described (Alexander, Schwartz, Balber, & Bangs, 2002 (link); Silverman et al., 2013 (link)). Pulse times: 2 min for VSG, 10 min for TbCatL and 15 min for p67. Chase times are specified in the relevant figures. Immunoprecipitated proteins were analyzed by SDS-PAGE followed by phosphorimaging of dried gels using a Molecular Dynamics Typhoon system with native ImageQuant Software (GE Healthcare, Piscataway, NJ).
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Metabolic Radiolabeling and Immunoprecipitation
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Corresponding Organization : University at Buffalo, State University of New York
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Variable analysis
independent variables
- Pulse times for radiolabelling with [35S]methionine/cysteine
- Chase times
- Levels of radiolabeled proteins after immunoprecipitation
- Analyzed by SDS-PAGE and phosphorimaging
- Immunoprecipitation of specific radiolabeled proteins
- SDS-PAGE and phosphorimaging analysis
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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