Hrp conjugated antirabbit igg ab6721

Anti-TOP1 antibody [EPR5375] (ab109374) was purchased from Abcam. Anti-TDP2 antibody (sc-377280) was obtained from Santa Cruz, and anti-GAPDH antibody (60004-1-lg) was obtained from Proteintech. Horseradish peroxidase (HRP)-conjugated antimouse IgG (ab6789) and HRP-conjugated antirabbit IgG (ab6721) antibodies were purchased from Abcam. Anti-TOP1cc (clone 1.1A; MABE1084) was purchased from Merck. CPT and ETP were obtained from Wako. TDP1 recombinant protein with an N-terminal GST tag (ab131921) was purchased from Abcam. ABC (A2694), AZT (A2052), Ara-C (C2035), and gemcitabine (G0544) were purchased from Tokyo Chemical Industry.

Samples of 10 μM pR248W, in the absence or presence of an equimolar amount of the ligands (ADH-1 or ADH-6), were incubated for various durations. The samples were then applied to a nitrocellulose membrane and dried for 1 h at room temperature or overnight at 4 °C. The membranes were subsequently blocked with 5% nonfat milk in TBST buffer (10 mM Tris, 0.15 M NaCl, pH 7.4, supplemented with 0.1% Tween 20) for 2 h at room temperature, washed thrice with TBST buffer, and incubated overnight at 4 °C with the polyclonal A11 antibody (AHB0052; Thermo Fisher Scientific) (1:1000 dilution in 5% nonfat milk in TBST buffer). Next, the samples were washed with TBST buffer (×3) and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (ab6721; Abcam, Cambridge UK) secondary antibody (1:500 dilution in 5% nonfat free milk in TBST buffer) for 1 h at room temperature. The dot blots were then washed with TBST buffer (×5), developed using the ECL reagent kit (Amersham, Piscataway, NJ), and finally imaged using a Typhoon FLA 9000 instrument (GE Healthcare Life Sciences, Pittsburgh, PA) with the settings for chemiluminescence.