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Mucin sialylation and characterization.

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posted on 08.04.2016, 23:05 by Andrés B. Lantos, Giannina Carlevaro, Beatriz Araoz, Pablo Ruiz Diaz, María de los Milagros Camara, Carlos A. Buscaglia, Mariano Bossi, Hai Yu, Xi Chen, Carolyn R. Bertozzi, Juan Mucci, Oscar Campetella

A) Mucins (or any glycoconjugate bearing a terminal β-galactose) may be tagged by the TS using a Neu5Az donor such as Neu5Azα(2–3)LacβOMe. Then the Azide group may be coupled via the Staudinger-Bertozzi chemistry or the Cu2+-free click chemistry to obtain a FLAG or biotin tag ready for detection. B) Western blot of Neu5Az trans-sialylated trypomastigote lysates revealing the relative molecular mass distribution of acceptor molecules. A line profile of the blot is also plotted. Neg Ctrl: Negative control. C) Neu5Az trans-sialylated parasites were submitted to organic solvents extraction as described in [20] to determine their mucin nature. Extracted material was subjected to Western blot. F1, F2 and F3 refer to the different purification fractions (for details see M&M). D) Neu5Az trans-sialylated trypomastigotes (900x106) were lysed and sialylated proteins pulled-down with anti-FLAG antibodies. Western blots of this material were revealed with anti-TcMUC II antibodies. E-F) Confocal images displaying partial colocalization of anti-FLAG and anti-TcMUC II (E) or anti-αGal (F) labeling at the parasite surface.