Cell Signaling Technology (CST)
Phospho-Histone H2A.X (Ser139)
#80312
D7T2V
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The antibody was used to detect the phosphorilation of the Ser139 following the addition of a chemical compound, thus evaluating DNA damage events
Western Blot
Hela cells, WCL
1:1000 milk 5% in PBS 0,05% tween over night
Milk 5% in PBS 0,05% tween
mouse 1:3000 milk 5% in PBS 0,05% tween 45 minutes
none
ECL
This antibody is very clean and without unspecific bands. Moreover, the signal is very strong when used following the CST datasheet instructions. Hela cells WCL has been loaded on a 4-12% SDS-precast gel (1:15h; 180 V) then proteins have been transferred on nitrocellulose membrane using the semi-dry blotting (1h 11V). Blocking with milk 5% in PBS 0,05% tween (1 hour) was followed by incubation overnight at 4 degrees (1:1000 in milk 5% in PBS 0,05% tween) Then the membrane was washed with 3x 5 minutes with PBS 0,05% tween, followed by a 1-hour incubation with Mouse secondary antibody 1:3000 in milk 5% in PBS 0,05% tween.To wash out the secondary antibody, a second wash with PBS 0,05% tween (3x5 minutes) was performed. ECL substrate has been used to detect the signal.***The CST data sheet suggests to use BSA to dilute the antibody (which is usually common for antibodies recognizing Phosphorlaiton sites) , but I used milk and it worked anyway.
High quality
None
Very Good