Purification and identification of HMGB1 secreted by liver cells and immune cells / 中南大学学报(医学版)

ABSTRACT

OBJECTIVE@#To purify and identify HMGB1 secreted by liver cells HepG2 and immune cells U937.@*METHODS@#We cultured the liver cell lines HepG2 and immune cell lines U937, and stimulated them with HMGB1 (400 ng/mL) for 20 h. Then the supernatant was collected. Ultrafiltration centrifugation, CM-Sepharose cation, DEAE-Sepharose anion exchange chromatography, Sephadex G75-gel filtration chromatography, and immunoprecipitation were used for purification. The molecular weight and identity of HMGB1 was confirmed by SDS-PAGE and Western blot.@*RESULTS@#A sharp stained protein band with a molecular weight of about 26 kD was obtained by SDS-PAGE analysis and shown to be HMGB1 confirmed by Western blot.@*CONCLUSION@#High purified HMGB1 can be separated from these two cell lines.