Pulse-Chase Analysis of Trypanosoma brucei

TbVps41 RNAi trypanosomes were either untreated or treated with 1 μg/ml tetracycline and used for pulse chase analysis. Pulse/chase analyses were performed using metabolic radiolabeling with [³⁵S]methionine/cysteine (Perkin Elmer) and subsequent immunoprecipitation of specific radiolabeled proteins from cell lysates as described previously (35 (link), 36 (link)). For p67, pulse time was 15 min and for TbCATL pulse time was 10 min. Chase times are as indicated in the relevant figures. Immunoprecipitated proteins were analyzed by SDS-PAGE and phosphorimaging using a Typhoon FLA 9000 with native ImageQuant Software (GE Healthcare).

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Ramakrishnan S., Baptista R.P., Asady B., Huang G, & Docampo R. (2021). TbVps41 regulates trafficking of endocytic but not biosynthetic cargo to lysosomes of bloodstream forms of Trypanosoma brucei. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 35(6), e21641.

Publication 2021

[35S]methionine/cysteine Typhoon FLA 9000 native ImageQuant Software

Cell Cysteine Immunoprecipitation Methionine Proteins Pulse Rnai Sds page Tetracycline Trypanosomes Typhoon

Corresponding Organization : University of Georgia

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Variable analysis

independent variables

Presence or absence of 1 μg/ml tetracycline treatment

dependent variables

Pulse/chase analysis of protein levels for p67 and TbCATL

control variables

Metabolic radiolabeling with [35S]methionine/cysteine
Immunoprecipitation of specific radiolabeled proteins from cell lysates
SDS-PAGE and phosphorimaging analysis of immunoprecipitated proteins

controls

Untreated (no tetracycline) condition as a negative control

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