Anti rat igg alkaline phosphatase

Protein samples (~1 mg) were isolated from the dorsal part of the thorax. Western blotting was carried out by using an SDS-containing gradient acrylamide gel, nitrocellulose blotting membrane, and TBST washing solution. In addition, 3% milk powder solved in TBST was used. Proteins were labeled at 4 °C overnight by the following primary antibodies: anti-ubiquitin (mouse, 1:500, Merck, Rahway, NJ, USA, ST1200), anti-Atg8a (rabbit, 1:2000 [64 (link),65 (link)]), anti-p62-vel (rabbit, 1:2000 [56 (link)]), anti-α-Tub84B (mouse, 1:2500, Sigma, St. Louis, MO, USA, T6199) and anti-EDTP (rat, 1:500 [19 (link)]). The following secondary antibodies were used (at room temperature for an hour): anti-rabbit IgG alkaline phosphatase (1:1000, Sigma, A3687), anti-mouse IgG alkaline phosphatase (1:1000, Sigma, A8438), and anti-rat IgG alkaline phosphatase (1:1000, Sigma, A8438). Primary and secondary antibodies were washed out 3 times for 10 min in TBST, and finally, membranes were incubated in an AP buffer. NBT/Bcip (Sigma, 72091) was used for recording the antibody staining, and NBT/Bcip was dissolved in an AP buffer (1:50).

Western blot samples were prepared from 10 female heads, which were treated in 32 μl of Fly Lysis buffer + 32 μl 2 × Laemmli buffer. 15 µl samples were run on 4–20% Mini-PROTEAN^® TGX™ Gel and blotted onto Nitrocellulose Membrane (Kisker Biotech, 40520100). After blocking with 3% Milk Powder (BioRad 170-6404 /Blotting-Grade Blocker/) dissolved in TBST, membranes were probed with specific antibodies [anti-Tubulin (1:1000, mouse, Sigma T6199), anti-Ref(2)P (1:2000, rabbit^{48 (link)}), anti-Atg8a (1:2500, rabbit^{50 (link)}), anti-EDTP, 1:1000, rat^{22 (link)}, anti-mouse IgG alkaline phosphatase (1:1000, Sigma, A8438), and anti-rabbit IgG alkaline phosphatase (1:1000, Sigma, A3687), anti-rat IgG alkaline phosphatase (1:1000, Sigma, A5153), and developed by NBT-BCIP solution (Sigma, 72091). Each western blot analysis was repeated at least three times with independent biological samples.