Preliminary constructing of lipopolysaccharide/lipopolysaccharide complexes

Objective To construct lipopolysaccharide/lipopolysaccharide binding protein(LPS/LBP) complexes by incubation for further study of LPS inflammatory signaling pathway. Methods LPS and LBP were mixed at following ratio: 15︰1, 10︰1 and 5︰1. The LPS/LBP mixtures, LBP (200 μg/ml, 100 μg/ml, 50 μg/ml) and LPS (100 μg/ml, 50 μg/ml) were incubated in constant temperature shaker overnight. The LPS/LBP mixtures, LBP and LPS were separate by gel electrophoresis. All the bands were harvested after coomassie blue staining for protein recovery, and endotoxin test. Results At relative molecular mass 52×103, LPS/LBP mixture groups and LBP groups appeared Coomassie blue staining bands, LPS groups didn′t show it. The higher concentration of LBP had the darker band . The endotoxin tests of all LPS/LBP mixture groups were positive. The endotoxin concentration of 15︰1 and 10︰1 LPS/LBP mixture groups were significantly higher than that of 5︰1 LPS/LBP mixture group(P 0.1). The endotoxin tests of all LBP groups were negative. LPS groups aslo showed negative results for endotoxin test. Conclusions The ideal ratio of LPS︰LBP to construct LPS/LBP complex is 10︰1. Purified LPS/LBP complex can be obtained by incubation and gel electrophoresis. Key words: Lipopolysaccharide; Lipopolysaccharide binding protein; Complexes; Construction