Recombinant human tg2

For the TG assay of ATXN1[82Q], 1 μg of purified human ATXN1[82Q] was mixed with 250 ng of normal or boiled (95°C for 3 minutes) recombinant human TG2 (R&D Systems, 4376) with or without cystamine (Tocris, 4981, final concentration: 40 mM) or LDN-27219 (Tocris, 4602, final concentration: 2 mM) to inhibit Tg activity in TG assay buffer (25 mM Tris-HCl, 5 mM CaCl₂, pH 8) supplemented with protease inhibitor cocktail. For the TG assay of ATXN1 with different polyQ-length, 1 μg of purified human ATXN1[2Q], ATXN1[30Q], or ATXN1[82Q] was mixed with 250 ng of recombinant human TG2 in the TG assay buffer supplemented with protease inhibitor cocktail. Total volume of the reaction mixture was 50 μL, and the final concentration of urea was 300 mM. The reaction mixture was incubated at 30°C for an hour with gentle shaking. For terminating TG reaction, NuPAGE LDS sample buffer (4×) was added into reaction mixture and boiled at 95°C for 15 minutes. For Western blot, samples were cooled at room temperature, loaded onto 3%–8% Tris-acetate gels and steps were performed as described above.

The optic grating sensor technique, Enspire Multimode Plate Reader (Perkin Elmer, MA, USA), was used to evaluate in-vitro protein interactions. The readout of this assay measured changes in the index of refraction upon a binding event, indicated by a shift of wavelength of the incident light. Briefly, recombinant human paxillin (Biolegend, CA, USA) at a concentration of 0.15 µg/µL was immobilized on the amine-coupling surface of microplate at pH7.5 and incubated overnight at 4 °C. Next day, the non-binding paxillin was washed off with 30uL of pH7.5 PBS for three times. After adding 15 uL of pH7.5 PBS, the plate was read and and the spectral shift was reset arbitrarily to zero. Fifteen microliters of recombinant human TG-2 (R&D, Minnesota, USA), human beta-3 integrin (R&D MN, USA) or vinculin (Abnova, CA, USA) at concentrations ranging from 0.0067 to 0.1 µg/µL was added to the respective wells of the microplate. This is then followed by incubation of the microplate in the Enspire Multimode Plate Reader at 27 °C for 2 hrs. The microplate was washed three times with PBS and then the spectral shift was measured and recorded. In the scenario where there was no significant interaction of the added protein with immobilised paxillin, 15 µL of a third recombinant protein was added into each well and the assay repeated. The above experiments were also repeated at pH 4.3.