Cell signaling technology (CST)
anti-P53
#2524
1C12
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The P53 antibody has been used in HEK293 cells do detect the endogenous P53. Despite the strong signal obatined, the antibody needs some small optimizations
Western Blot
HEK293 WCL
1:1000 overnight
Milk 5% in PBS 0,05% tween
mouse 1:3000 milk 5% in PBS 0,05% tween 45 minutes
None
ECL
This antibody has a strong background if not optimazed, but has not unspecific bands. The dilution of 1:1000 reported on the CST datasheet is an optimal condition.HEK293 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 (45h 12V). Blocking with milk 5% in PBS 0,05% tween (1 hour, but maybe even 30 minutes longer is better), was followed by incubation over night with P53 antibody (1:1000 in milk 5% in PBS 0,05% tween) Then the membrane was washed with 3x 10 minutes with PBS 0,05% tween, followed by a 1-hour incubation with Mouse secondary antibody 1:3000 in milk 1% in PBS 0,05% tween. To wash out the secondary antibody, a second extensive wash with PBS 0,05% tween (3x 10 minutes) was performed. ECL substrate has been used to detect the signal.
Very nice signal and very sensitive to increase-decrease of the p53 levels after drug treatment
Strong background
I would raccomand this product, easy to use and does its job.