4.2.5. TEM Analysis

Ub2(48)tau4RD(353) sample was further purified by HPLC-reverse phase C18 done with TFA 0.1%/acetonitrile gradient, before buffer exchange to the aggregation buffer.

Samples of Ub2(48/63)tau4RD(353) conjugates, tau4RDΔC, and Ub-tau4RD(353) were incubated at concentration of 0.05 mM in 20 mM sodium phosphate buffer at pH 7.4 and 50 mM NaCl (with 0.02% NaN3 and protease inhibitors with EDTA) at 37 ◦C without agitation, with the addition of equimolar amount of heparin as aggregation initiator. After 48 h of incubation, we used 100 kDa cut-off filters (Sartorius, Aubagne, FR) to exchange the buffer to H2O mQ.

For TEM measurements, we used a Tecnai G<sup>2</sup> (FEI, Hillsboro, OR; USA) transmission electron microscope operating at 100 kV, with the procedure described in [30]. Images were analyzed with the ImageJ software.
