Hrp conjugated goat anti mouse igg

Ovary protein lysates were obtained from w¹¹¹⁸; GFP-Mxc, w¹¹¹⁸; GFP-Mxc^LisH-AAA, and w¹¹¹⁸; GFP-Mxc^SIF-AAA female flies dissected in 1× Tris PBS. Ovaries were snap-frozen in dry ice and ethanol for 10 min and stored at −20°C overnight. Ovaries were resuspended in buffer containing 4% SDS and dissociated with 20 strokes of a Dounce homogenizer on ice. Equal amounts of protein were run on a 7.5% acrylamide gel (Bio-Rad, Hercules, CA) and then transferred to a polyvinylidene fluoride membrane (Millipore) presoaked in methanol for 15 min at room temperature. Membranes were incubated overnight at 4°C in primary rabbit anti-GFP (Abcam) antibody to detect GFP-tagged proteins (∼250 kDa in size) and in primary mouse anti-lamin (Developmental Studies Hybridoma Bank) antibody to detect Drosophila lamin (74 kDa in size). Lamin and an anti-GFP antibody cross-reacting band were used as loading controls. Subsequently membranes were incubated in secondary antibody horseradish peroxidase (HRP)–conjugated donkey-anti rabbit IgG (GE Healthcare Life Sciences) and HRP-conjugated goat-anti mouse IgG (GE Healthcare) for 2 h at room temperature to detect GFP and lamin, respectively. The signal was enhanced using Enhance Signal West Dura (Thermo Scientific, Rockford, IL) and visualized using an 8-MP EX Sigma camera in a BioSpectrum imaging system (UVP) after a 25-min exposure.

Whole cell extract was prepared as described previously [3] (link), and 20 μg of whole cell extract was subjected to 7.5% SDS–PAGE. Proteins were transferred to a PVDF membrane in 48 mM Tris, 39 mM glycine, 0.025% SDS and 15% methanol by wet transfer. The membrane was blocked in 5% skim milk and incubated with a rabbit polyclonal anti-GFP antibody (final concentration 0.1 μg/mL; Rockland, USA) or a mouse monoclonal anti-Nup98 antibody, 13C2 (final concentration 0.3 μg/mL) [20] (link). HRP-conjugated goat anti rabbit IgG or HRP-conjugated goat anti mouse IgG (GE Healthcare, USA) was used as a secondary antibody. For a loading control, endogenous actin was detected on the same membranes after stripping using a mouse monoclonal anti-β-actin antibody, ab8224 (final concentration 0.1 μg/mL; Abcam, USA) and HRP-conjugated goat anti-mouse IgG as described above. Protein bands were detected by chemiluminescence using the ChemiDoc MP imaging system (Bio-Rad, USA).