Proteins from cell extracts were resolved by SDS-PAGE on a 4-20% Tris glycine gel and were transferred to PVDF membrane. Membranes were incubated with either 1 µg/mL anti-PARP1 (pan) antibody or anti-PARP1 cleavage site (214/215) specific antibody at 1 µg/mL. After washing, membranes were incubated with goat F(ab’)2 antirabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar detection method. The data show that the anti-PARP1 cleavage site specific antibody only recognizes the 85 kDa fragment of PARP1 in apoptotic cells (lane 3) and does not react with full length PARP1 (lane 1). The PARP1 (pan) antibody confirms that nonapoptotic cells express full length PARP1 of 116 kDa (lane 2) and which is cleaved when apoptosis is induced (lane 4).

HeLa cells were induced into apoptosis with staurosporine at 0.5 µM for 5 hours (panel A) and were untreated as control (panel B). The cells were fixed in cold acetone for 5 minutes. Cells were incubated with the anti-PARP1 cleavage site specific antibody (CCSA) at 10 µg/mL. Cells were then incubated with biotinylated goat anti-rabbit Igs followed by ABC and DAB. The data show that the anti-PARP1 CCSA specifically recognizes PARP1 in apoptotic cells. Taken together with Western blot data above, these data demonstrate the specificity of the anti-PARP1 CCSA for cleaved PARP1.

See Abreview