Mucin sialylation and characterization.
2016-04-08T23:05:59Z (GMT) by
<p>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 Cu<sup>2+</sup>-free click chemistry to obtain a FLAG or biotin tag ready for detection. B) Western blot of Neu5Az <i>trans</i>-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 <i>trans</i>-sialylated parasites were submitted to organic solvents extraction as described in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005559#ppat.1005559.ref020" target="_blank">20</a>] 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005559#sec010" target="_blank">M&M</a>). D) Neu5Az <i>trans</i>-sialylated trypomastigotes (900x10<sup>6</sup>) 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.</p>