Detection of Newly Synthesized Proteins via Metabolic Incorporation of Non-natural Amino Acid

Abstract 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-α). Experimental parameters including the concentration of LPS and the duration of LPS treatment were investigated by measuring the LPS-stimulated TNF-α 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 alkyne-azide cycloaddition (CuAAC). Then TNF-α 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.