Abcam
Anti-6X His tag antibody
ab84162
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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.
Western Blot
MCF-7 cells
4 degrees Celsius overnight
5% milk
Donkey anti-sheep IgG HRP conjugate
1/10000 at room temperature for 1 hour
ECL
We co-transfected His-tagged ubiquitin and HA-tagged Rad17 intoMF-7 cells and collected cells after exposure to UV. UV-induced ubiquitylation of Rad17 was measured by immunoprecipitation of Rad17 by anti-HA antibody coupled with immunoblottingusing anti-His antibody. As indicated in the Figure, the levels of ubiquitin conjugated Rad17 elevated following exposure to UV radiation in the presence of MG132.
None
It works well for blotting.
NA
It works as expected.