Detoxi gel endotoxin remover resin

Recombinant human Hsp70 was purified from E. coli (strain BL21 DE3), transformed with a pMS-Hsp70 plasmid, as previously described^{20 (link)}. The Hsp70 solution was further detoxified by incubation with Detoxi-Gel Endotoxin Remover Resin (Thermo Scientific, USA) and sterilized by filtration through a 0.22 µm filter (Millipore, USA). According to the E-Toxate assay (Sigma-Aldrich, USA), the level of lipopolysaccharide in the final Hsp70 preparation was lower than 0.25 U/mL. For flow cytometry and confocal experiments, Hsp70 was labeled with Alexa-555 or Alexa-488 or with NHS–biotin (Sigma, USA), according to manufacturer’s instructions. To remove unbound dye (or non-reacted biotin) after labeling the protein was subjected to the dialysis: twice at room temperature for 1 h, and then at 4 °C overnight, stirring.