Colloidal coomassie blue g 250

Dissected glands or brains were homogenized in 100 µl of SDS-PAGE loading buffer and boiled for 5 min. Appropriate amounts of extracts (between 0.2 and 6.0 brain and HG equivalent, respectively) were loaded on a vertical 10% SDS-PAGE gel and electrophoresed at 15 V/cm (horizontal gel system, PeqLab, Erlangen, Germany). Obtained gels were either stained with colloidal Coomassie blue G-250 (Sigma, St Louis, USA) or blotted onto nitrocellulose membrane (semidry system, 2 V/cm², PeqLab). Blotting membranes were blocked overnight with 5% skimmed milk in Tris-buffered saline with Tween 20 TBST (10 mM Tris, pH = 7.4, 150 mM NaCl, 0.05% Tween 20). Primary antibodies were diluted in TBST as follows: rabbit affinity-purified α-BtRJPL (Kupke et al., 2012 (link)) 1:1,000, and goat α-actin (Santa Cruz, San Diego, USA) 1:500. Incubation varied from 3 hours to overnight. After washing 4×10 min with TBST, the blots were incubated for 1 hour with fluorescence-labeled secondary antibodies (anti-goat 680 and anti-rabbit 800; LI-COR Biosciences, USA) diluted 1:20,000 in TBST. After final washing 4×10 min with TBST, immunoreactive bands were detected by an Odyssey infrared imaging system (LI-COR Biosciences, USA).

For the identification of proteins in spots of interest, a separate preparative two-dimensional gel electrophoresis was performed using a total of 800 µg proteins per gel. Resolved protein spots were visualized by Colloidal Coomassie Blue G-250 (Merck, Darmstadt, Germany).