Alexa fluor 680 donkey anti goat

Wandering third instar VNCs from 20 larvae were homogenized in 100 μL of lysis buffer (20 mM HEPES, 10 mM EDTA, 100 mM KCl, 0.1% (v/v) Triton X-100, 5% (v/v) glycerol) with a protease inhibitor cocktail (Roche, 04693132001) combined with a protease and phosphatase inhibitor cocktail (Abcam, ab201119). All samples were then sonicated and run in 4% to 15% Mini-PROTEAN TGX Stain-Free Precast Gels (BioRad, 4568083) alongside Precision Plus Protein all blue prestained protein standards (BioRad, 1610373). Next, total protein was transferred to PVDF membranes using a Trans-Blot Turbo system (BioRad). After transfer, the membrane was blocked by TBS intercept blocking buffer (LiCOR, 927–60000) for 1 hour at RT. The blocked membranes were incubated with primary antibodies overnight at 4°C. Antibodies used include rabbit anti-Csw (Lizabeth Perkins, F1088, 1:500) and goat anti-GAPDH (Abcam, ab157157, 1:2,000). The membrane was washed with Tris-buffer saline with 0.1% Tween-20 (TBST) and then incubated with secondary antibodies for 40 minutes at RT. Secondary antibodies used include Alexa Fluor 680 donkey anti-goat (Invitrogen, A21084, 1:10,000) and Alexa Fluor 800 goat anti-rabbit (Invitrogen, A32735, 1:10,000). After washing with TBST (3X, 10 minutes), the membranes were imaged using the Li-COR Odyssey CLx system.