Anti-Cleaved PARP1 antibody [4B5BD2] (ab110315)

Lane 1: Wild type HAP1 (untreated) whole cell lysate (20 µg)
Lane 2: PARP1 (untreated) knockout HAP1 (untreated) whole cell lysate (20 µg)
Lane 3: HeLa (untreated) whole cell lysate (20 µg)
Lane 4: HAP1 (staurosporine treated, 1 uM, 4 hr) whole cell lysate (20 µg)
Lane 5: PARP1 (staurosporine treated, 1 uM, 4 hr) knockout HAP1 whole cell lysate (20 µg)
Lane 6: HeLa (staurosporine treated, 1 uM, 4 hr) whole cell lysate (20 µg)

Lanes 1 - 6: Merged signal (red and green). Green - ab110315 observed at 100 kDa. Red - loading control, ab181602, observed at 37 kDa

ab110315 detected the expected band for cleaved PARP1 in wild type HAP1 cells treated with staurosporine and the band was not seen in PARP1 knockout cells treated with staurosporine. Wild-type and PARP1 knockout samples were subjected to SDS-PAGE. ab110315 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/10000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Immunocytochemistry images of stained untreated (A) and 4 hours 1 µM Staurosporine-treated (B) Human HeLa cells. The cells were paraformaldehyde fixed (4%, 20 minutes) and Triton X-100 permeabilized (0.1%, 15 minutes). The cells were incubated with 1.0 µg/ml ab110315 for 2 hours at room temperature or over night at 4°C. 10% goat serum was used as the blocking agent for all blocking steps. The secondary antibody was Alexa Fluor^® 488 goat anti-mouse IgG (H+L) (in green) used at 2.0 µg/ml for 2 hours. DAPI was used to stain the cell nuclei (in red). Heat induced antigen retrieval (0.1 M Tris-HCl, 5% urea, pH 9.5 for 5 min at 95°C) improves signal. Note that the ab110315 labels only condensed and/or fragmented nuclei of apoptotic Staurosporine-treated cells.

Western Blot analysis using ab110315 antibody and 20 µg of untreated (CON) or 4 hours 1 µM Staurosporine-treated (STS) HeLa cells. Blots were incubated with an antibody that recognizes both the full-length PARP1 and its 89 kDa fragment (left panel), or 1.0 µg/mL PARP1 (cleaved) antibody (ab110315) (right panel). Appropriate HRP-conjugated secondary antibodies followed by ECL detection were used. Note that the MS777 antibody recognizes the apoptosis-specific 89 kDa fragment of PARP1 but it does not recognize the full-length PARP1.