Cell signaling technology (CST)
Parp antibody
#9542
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PARP antibody has been used to detect the activation of apoptosis related pahtways followinf treatment with two compounds.
Western Blot
Hela cells, cell lysate
1:1000 milk 5% in PBS 0,05% tween
Milk 5% in PBS 0,05% tween
Rabbit 1:3000 milk 5% in PBS 0,05% tween 1 hour
none
ECL
WCL of HelaS3 treated with DMSO (control) and two different drugs has been collected and 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 1h at RT with GAPDH antibody (1:3000 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 Rabbit secondary antibody 1:3000 in milk 5% 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.The antibody can be dirty and the detection of the cleaved band results to be tricky. The molecular weight of the full PARP and cleaved PARP are slightly higher than expected.
Good signal for the PARP full length.
Bad signal for the cleaved band and the the molecular weight can be different from the expected one.
This antibody has potential, but needs, for sure, further optimizations.