Journal: Immunology and Cell Biology

Article Title: A hybrid protein is a functional molecule to reduce the cytokine storm caused by excessively activated macrophages

doi: 10.1111/imcb.70000

Figure Lengend Snippet: Design of a hybrid protein (hMIKO‐1) and its purification. (a) The frames of the full‐length AA sequences of hS100A8 and hS100A9 were schematically drawn by blue and gray horizontal bars, respectively. hMIKO‐1 is a hybrid protein of hS100A8 and hS100A9. The whole frame of hMIKO‐1 was as follows: 21 AA residues from the C terminus of hS100A9 were added to the C terminus of hS100A8. (b) SDS–PAGE (15% gel) was performed to confirm the purity of hMIKO‐1 in the absence of 2‐mercaptoethanol. hMIKO‐1 was purified by affinity chromatography (Ni‐agarose; Nacalai Tesque, Kyoto, Japan), ion‐exchange chromatography (DEAE cellulose; Bio‐Rad Co., Ltd, USA) and gel filtration (Sephacryl S‐300 HR). Proteins in the gel were stained with Coomassie Brilliant Blue. Lanes M and 1 were molecular markers and purified hMIKO‐1, respectively. As shown in the kit (Toxinsensor, Endotoxin Detection System; GenScript Inc., USA), the concentration of contaminated endotoxin present in the product hMIKO‐1 was less than 0.3 EU mg −1 protein. This result indicates no significant effects in subsequent experiments (data not shown). AA, amino acid; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.

Article Snippet: Contaminated endotoxin was deleted as much as possible using a DEAE cellulose (Macro‐Prep High Q Media, Bio‐Rad, USA) column [26 mm (D) × 80 mm (L)].

Techniques: Purification, SDS Page, Affinity Chromatography, Ion Exchange Chromatography, Filtration, Staining, Concentration Assay, Polyacrylamide Gel Electrophoresis