Cell Signaling Technology (CST)
GAPDH
#2118
(14C10)
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GAPDH antibody has been used to normalize Western Blot results in Hela cells. Hela cells have been treated with different concentrations of chemical compounds, nevertheless, the GAPDH signal is stable and not affected.
Western Blot
Hela cells, WCL
1:3000 milk 5% in PBS 0,05% tween
Milk 5% in PBS 0,05% tween
Rabbit 1:3000 milk 1% in PBS 0,05% tween
None
ECL
This antibody is very clean and without unspecific bands. Moreover, the signal is very strong when used following the CST datasheet instructions. The dilution of 1:1000 is risky to use since can easily burn the membrane; on the other hand, a dilution of 1:3000 gives very good results even with 30 minutes or 1-hour incubation at room temperature, shortening times and saving antibodies. 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 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 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.
The signal is very clean and clear.
Following the datasheet instructions there is the risk to burn the membrane. Its better to use a lower concentration.
I strongly recommend this antibody for clean and fast results.