Mini dialysis devices

Photo-oxidation experiments were performed as described previously [26 (link)] with minor modifications. In brief, solutions containing disulfide-containing proteins (aLA, LYSO, CRP and B2M, in 10 mM phosphate buffer, pH 7.4) and Rose Bengal (RB, final concentration 10 μM) were exposed to the light from a Leica P 150 slide projector through a 345-nm cut-off filter. After photolysis, samples were treated with catalase (1 mg mL⁻¹) to remove H₂O₂ generated during the photo-oxidation which might also react with the added thiol-containing proteins. Catalase was not added before the photolysis as this enzyme has a significant optical absorption, and therefore interferes with the photosensitization reaction, as well as acting as a competitive target for ¹O₂. In experiments where the role of the disulfide bond in B2M was examined, a combination of DTT and NEM (both 100-fold molar excess over the protein; in 10 mM phosphate buffer, pH 7.4, reaction for 1 h at 21 ^oC) were used to reduce and alkylate the disulfide bonds of B2M (20 μM), with excess DTT and NEM subsequently removed using mini dialysis devices (3.5 kDa molecular mass cut-off; ThermoFisher) and 10 mM phosphate buffer, pH 7.4, prior to photo-oxidation.