ABSTRACT: To purify and identify HMGB1 secreted by liver cells HepG2 and immune cells U937.
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.
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.
High purified HMGB1 can be separated from these two cell lines.
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences 11/2011; 36(11):1065-70.