Anti-6X His tag antibody

MCF7 cells were transfected with HA-Rad17, and His-Ubquitin prior to UV-irradiation. The next day culture medium was removed and plates were washed twice with 1×PBS. Then the plates were irradiated with 10J UV without the lids. Medium with or without MG132 was added for further incubation. Cells were prepared in lysis buffer (25 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.5% Nodidet P-40, 1 mM EDTA, 5 mM MaF and 1× protein inhibitor cocktail) on ice for 30 minutes. Then 27G1/2 syringes were used to shred the DNA. The supernatants were collected after centrifugation at 12000g for 30 minutes. Equal amount of protein lysates were aliquoted, and equal amount of primary antibody was added to the above lysates. After rotation at 4°C overnight, equal amount of immobilized protein A/G beads (Pierce, Rockford, IL) were added to the tubes. After rotation again at 4°C for 4 hours, the beads were collected by centrifugation at 2500 g for 3 minutes. Electrophoresis loading buffer was added to the beads after washing with IP wash buffer (25 mM Tris-HCl, pH7.5, 150 mM NaCl, and 1× protein inhibitor cocktail) 5 times. After denaturing at 95°C for 5 minutes, the supernatants were separated and transferred to nitrocellulose membranes. After blocking, the membranes were incubated with the appropriate primary antibody at 4°C overnight. After washing, the membranes were incubated with secondary antibody at room temperature for 1 hour. Proteins were detected with the enhanced chemiluminescence (ECL) kit (GE Healthcare, Piscataway, NJ).

We used several His antibodies to detect the signal, but they didn't work. In the end we chose this one because Abcam had a very good reputation for product quality although the price is high. Fortunately it worked very well.