A method for the detection of newly synthesized protein via metabolic incorporation of non-natural amino acid was developed. Raw264.7 cells were treated with lipopolysaccharide (LPS) to generate tumor necrosis factor alpha (TNF-alpha). Experimental parameters including the concentration of LPS and the duration of LPS treatment were investigated by measuring the LPS-stimulated TNF-a from Raw264.7 cells. The optimal experimental conditions were determined as follows. Raw264.7 cells were cultured in Met-free DMEM containing 1% fetal bovine serum (FBS), and then treated for 4 h with 10 ng mL(-1) LPS in presence of azidohomoalanine (AHA). The biotin tags were introduced into proteins containing AHA by copper-catalyzed allcyne-azide cycloaddition (CuAAC). Then TNF-alpha from was specifically captured by the antibody adsorbed on the solid support, on which the biotin tag could be detected by streptavidin coupled with horseradish peroxidase (HRP). This study provided a method for the detection of a specific newly synthesized protein, and characterization of specific protein turnover.
[1]Jilin Univ, Hosp 2, Cent Lab, Key Lab Zoonosis,Minist Educ, Changchun 130041, Jilin, Peoples R China.
[2]Jilin Univ, Hosp 1, Dept Endocrinol, Changchun 130012, Jilin, Peoples R China.
[3]China Japan Union Hosp, Dept Neurol, Changchun 130033, Jilin, Peoples R China.
(8.0+极速模式)