4.2.3. Disulfide-Coupling Reaction

First, Ub2(48/63)-SH chains were activated with 5,5--Dithiobis(2-nitrobenzoic acid) (DTNB). Typically, 4-6 mg of DTNB were dissolved in 3 mL of 100 mM Hepes bu ffer pH 7.0 containing Ub2-SH at a concentration of 1-2 mg/mL. After incubation at 10 ◦C overnight, the obtained Ub2(48/63)-S-(2-nitro-5-thiobenzoic acid) (hereafter Ub2(48/63)-S-TNB) disulfide adducts were purified by desalting. The activated proteins were verified by MALDI (Figure 4a,b).

Then, the Ub2(48/63)-S-TNB disulfide adducts were incubated with tau4RD(353) in equimolar amount in 100 mM Hepes bu ffer pH 7.0, for 20' at 25 ◦C. The Ub2(48/63)tau4RD(353) disulfide conjugates were then purified by SP-ion exchange chromatography and verified by MALDI (Figure 4c,d).

It is important to note the thiol containing proteins Ub2(48/63)-SH and tau4RD(353) were first incubated with a large excess of DTT that was then properly removed by size exclusion chromatography just before the disulfide coupling reaction.

The procedure to obtain mono-ubiquitinated tau protein at position 353 (Ub-tau4RD(353)) by a similar disulfide-coupling strategy is described in [30].
