Hrp conjugated goat anti guinea pig

Ovaries were dissected in Grace’s medium and processed for IP as described previously [16 (link)]. For western blotting, one-half ovary equivalent lysate was loaded into each lane of an 8% or 10% SDS-PAGE. The following primary antibodies were used: mouse anti-Aub (1:1,000, from Dr Siomi), mouse anti-Ago3 (1:500, from Dr Siomi), rat anti-Tap (1:1,000, this study), mouse anti-c-Myc 9E10 (1:5000, Sigma), mouse anti-HA (1:5,000, Roche, BASEL, Switzerland), mouse anti-FLAG M2 and its horseradish peroxidase (HRP)-conjugated secondary antibody (1:1,000, Sigma), guinea pig anti-Vas (1:5,000) [16 (link)], rabbit anti-Piwi (1:500, Abcam, Cambridge, England, United Kingdom Ab5207), mouse anti-Piwi (1:50, from Dr Siomi), rabbit anti-SpnE (1:500, from Dr Dahua Chen) and mouse anti-α-Tubulin DM1A (1:1,000, Santa Cruz Biotechnology, Santa Cruz Biotechnology, Dallas, Texas, U.S.A.). Immunoreactive bands were visualized using HRP-conjugated goat anti-guinea pig (Dako, Dako North America, Inc. Carpinteria, CA, USA), anti-rabbit, anti-rat or anti-mouse secondary antibodies (Bio-Rad, Hercules, CA, United States of America) at 1:5,000, and developed with the SuperSignal West Pico Chemiluminescent Substrate detection reagent (Thermo Scientific).

Protein lysates from ovaries (one-half ovary equivalent per lane) were electrophoresed on a 7.5% or 10% sodium dodecyl sulfate-polyacrylamide gel. The following primary antibodies were used: guinea pig anti-dMarf1 (1:1,000, this study), mouse anti-Myc 9E10 (1:5000, Wako), mouse anti-CycA (1:1000, DSHB, IN, USA), mouse anti-CycB (1:1000, DSHB), rabbit anti-Nanos (1:1000, from Dr. Nakamura), rabbit anti-Dhd (1:1000, from Dr. Loppin), mouse anti-Polo (1:400, from Dr. Glover), mouse anti-glorund (1:1000, DSHB), and mouse anti-αTubulin DM1A (1:1000, Santa Cruz Biotechnology). Immunoreactive bands were visualized using HRP-conjugated goat anti-guinea pig (1:1000, Dako, Denmark), anti-rabbit, or anti-mouse secondary antibodies (1:3000, Bio-Rad, CA, USA), and immunoblots were detected using LAS-1000 (GE Healthcare) with an ECL chemiluminescent substrate (Thermo Fisher Scientific).