Ab97085

A sandwich ELISA was used to detect Poly(ADP-Ribose) (PAR) polymers. Cells were boiled in PathScan Sandwich ELISA Lysis Buffer (Cell Signaling Technology) supplemented with 1 mM phenylmethanesulfonyl fluoride (Sigma). Cell extracts were then diluted in Superblock buffer (Thermo Scientific) prior to the ELISA. A 96-well polystyrene plate (Thermo Scientific Pierce White Opaque) was coated with 100 μl per well carbonate buffer (1.5 g/l sodium carbonate Na₂CO₃, 3 g/l NaHCO₃) containing the capture antibody (mouse anti-PAR at 4 μg/ml, Trevigen 4335) overnight at 4°C, after which it was washed with PBST solution. The wells were then blocked with Superblock at 37°C for 1 hour. Then, 10 μl of cell extract was added to 65 μl of Superblock, applied to each well in triplicate, and incubated overnight at 4°C, after which it was washed with PBST solution. Then the detection antibody (Rabbit anti-PAR, Trevigen 4336, diluted 1/1000 in PBS/2% milk/1% mouse serum) was added and incubated for 1 hour at room temperature. After washing, secondary antibody HRP-conjugated anti-rabbit (Abcam, ab97085, diluted 1/5000 in PBS/2% milk/1% mouse serum) was applied to each well for 1 hour. To read out, 75 μl of substrate for the enzyme (Supersignal Pico, Pierce) was added to each well. The optical absorbance (OD λ = 425 nm) was recorded at various time points (1, 5, and 15 minutes).