Pierce ecl plus western blotting substrate systems

Adrenal glands and brain extracts were collected from P0-1 Syt-7 WT and KO mice and lysed in RIPA buffer supplemented with Protease Inhibitor Cocktail (Invitrogen, 89900). The supernatants were collected and protein concentrations were estimated by using the BCA Protein Assay Kit (Pierce 23227) and plotting the resulting BSA curve. 25 μg and 15 μg of protein, from adrenal glands and brain extracts, respectively, were resolved by 4–12% SDS-PAGE (Invitrogen, Thermo Fisher Scientific) and wet-transferred onto an Amersham Hybond LFP PVDF membrane (GE Healthcare). The membrane was blotted with rabbit polyclonal α-Syt7 (1:500; Synaptic Systems SY105173) and mouse monoclonal α-VCP (1:10000; Abcam ab11433), as a loading control, followed by HRP-conjugated α-rabbit (1:2000; Agilent Dako-P0448) and HRP-conjugated α-mouse (1:10000; Agilent Dako-P0447) secondary antibodies. The blot was developed by chemiluminescence SuperSignal West Femto and Pierce ECL Plus Western blotting substrate systems (Thermofisher Scientific) and immunoreactive bands were detected using the FluorChemE image acquisition system (ProteinSimple) equipped with a cooled CCD camera.

Extracts from HEK293FT cells expressing ubMunc13-2-EGFP or Munc13-1-EGFP, as well as non-expressing cells, were collected and lysed in RIPA buffer supplemented with Protease Inhibitor Cocktail (Invitrogen, 89900). The supernatants were collected and protein concentrations were estimated using the BCA Protein Assay Kit (Pierce 23227) after plotting the resulting BSA curve. 25 mg of protein was resolved by 4–12% SDS-PAGE (Invitrogen, Thermo Fisher Scientific) and wet-transferred onto an Amersham Hybond LFP PVDF membrane (GE Healthcare). The membrane was blotted with rabbit polyclonal α-GFP (1:300; Synaptic Systems SY132003) and HRP-conjugated mouse monoclonal α-β-Actin-Peroxidase (1:10000; Sigma-Aldrich A3854), as a loading control, followed by HRP-conjugated α-rabbit (1:2000; Agilent Dako-P0448) secondary antibody. The blot was developed by chemiluminescence Pierce ECL Plus Western blotting substrate systems (Thermofisher Scientific) and immunoreactive bands were detected using the FluorChemE image acquisition system (ProteinSimple) equipped with a cooled CCD camera.