The objective of the study was to determine if there was an interaction between USP7 and EBNA1. Prior to experiments, the DNA sequences of EBNA1 and its fusion tag - GST, USP7 and its corresponding fusion tag - 6xHis, as well as the GST tag alone were each inserted into three separate 10 ml cultures of E.coli BL21 using plasmids. Therefore, each culture expressed one of the peptides 6xHis-USP7, GST and GST-EBNA1. The GST-EBNA1 culture was placed in 45 ml of LB Amp+ medium, while 6xHis-USP7 and GST were mixed with 45 ml of LB Amp+ Km+ medium. Cultures were left in shaking incubators at 250 rpm for one hour at 37°C. An optical density reading was taken on 1 ml samples of each culture. Optical densities were within the ideal range of 0.8 to 1.0, indicating that sufficient protein had been expressed and no further incubation was necessary. 150 µL of GST-EBNA1 was removed from the cuvette and briefly centrifuged; the pellets were isolated at -20°C. Then, 0.55 ml of 0.1 M IPTG was added to each culture and placed in the shaking incubator at 37°C for one hour to cause the expression of the peptides. The induced cultures were centrifuged at 4°C for 20 min at 4000 rpm and the pellets were stored at -20°C until use. Lysis/binding buffer was prepared with 5 μL of 5 M imidazole and 5 ml of common buffer (500 mM NaCl and 50 mM Tris). HCL at pH7.5) and left on ice. 1 ml of this buffer was added to the thawed 6xHis-USP7 pellets. The mixture was vortexed, placed on ice, and lysed with a closed sonicator for four 20-second pulses at 15-second intervals, at 30% intensity. The refrigerated microcentrifuge was used for 10 min at 13,000 rpm to isolate insoluble proteins as well as unlysed cells. The supernatant was isolated and 10 µL of the lysate was middle of paper......xHis-USP7 was washed through the GST column and failed to bind the GST protein, producing a single band on the GST column. MSDS PAGE. The observed 6xHis-USP7 band is extremely weak, probably due to the fact that the volume of purified protein used was only 20 µL. Nevertheless, 6xHis-USP7 was not washed out in the GST-EBNA1 column and the GST-EBNA1 pull-down unbound protein sample did not yield any bands (Figure 4). Furthermore, neither the GST pull-down wash nor the GST-EBNA1 pull-down wash yielded bands. When washing the column with reduced glutathione-rich GST elution buffer, the GST pull-down eluent yielded a single thick band corresponding to the GST protein. GST-EBNA1 pull-down elution, however, yielded two distinct bands. The heavier band that traveled a shorter distance corresponded to GST-EBNA1, while the lighter band corresponded to 6xHis-USP7 (Figure 4).