Kinase experiment:

Binding experiments are performed at 37°C with T2-Db cells, with a Millipore MultiScreen assay system. The H-2Db LCMV antigen gp276-286 (SGVENPGGYCL) is radioiodinated, and the radiolabeled peptide is purified. Cells (2×105 per well) are incubated in MultiScreen-HV 96-well filtration plates (pore size, 0.45 mm) with 125I-gp276-286 (10 nM [final concentration]) for 90 min at 37°C. Cells are washed three times with ice-cold 1% BSA-PBS and by filtration under vacuum. The radioactivity bound to the cells retained on the filter is counted with a gamma counter. Direct binding is measured in the absence (total binding) or the presence (nonspecific binding) of a 1,000-fold excess (10 mM) of unlabeled gp276-286. Specific binding to H-2Db is defined as the difference between total binding and nonspecific binding. Nontransfected T2 cells are used as a negative control under the same experimental conditions. Competition assays are performed with increasing concentrations (10-10 to 10-5 M) of unlabeled peptides competing against a fixed concentration (10-8 M) of 125Igp276-286. The percent inhibition of binding is calculated as 100 × [1-(counts per minute in the presence of competitor - counts per minute of nonspecific binding)/counts per minute of specific binding].

参考文献:

[1]. Gairin JE, et al. Optimal lymphocytic choriomeningitis virus sequences restricted by H-2Db major histocompatibility complex class I molecules and presented to cytotoxic T lymphocytes. J Virol. 1995 Apr;69(4):2297-305.