Detection of NF-κB activity in T cells by electrophorestic mobility shift assay and enhanced chemiluminescent technique

Objective: To establish a method for detection of NF-κB activity in human T cells after activation using gelelectrophorestic mobility shift assay(EMSA) and enhanced chemiluminescent(ECL) technique.Methods: Human peripheral blood mononuclear cells(PBMC) were stimulated with anti-CD3 mAb to generate activated T cells.Cell nuclear protein extracts were mixed with biotin-label probe of NF-κB specific oligonucleotides sequence,and protein-probe complex were separated from free probe by electrophoresis in nondenaturing polyacrylamide gel,and electrotransferred to positive charged nylon membrane,and added with streptoavidin-HRP conjugate and enhanced chemiluminescent reagent to detect NF-κB activity.Results: The NF-κB shift band were detected at 15 min after stimulation of human PBMC,and reached maximum peak at 30 min,and then decreased at 60 min of stimulation,and the results of control group demonstrated the method with high specific and sensitivity.Conclusions: Enhanced chemiluminescence based EMSA is very convenient and high sensitive technique to detect nuclear factor such as NF-κB activity in T cells after activation.