Western lightning plus ecl detection kit

Cells were lysed in buffer containing 20 mM Tris (pH 7.4), 100 mM NaCl, 1 % Triton X-100, 1 mM MgCl, 1 mM EDTA, 10 % glycerol, and supplemented with phosphatase and protease inhibitors (Sigma-Aldrich). Protein concentrations were determined using a Bio-Rad DC Protein Assay Kit (Bio-Rad). Proteins were separated by polyacrylamide gel electrophoresis (TGX Stain-Free – Precast Gels), and transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were hybridized with primary antibodies against NAPRT: 40 ng/mL HPA023739 (Sigma-Aldrich), 100-200 ng/mL 3C6D2, 2 μg/mL CLO366, and 4 μg/mL each for antibodies 4A9 and 5B0 or anti-α-tulin antibody (DM1A, Sigma). Secondary antibodies were goat anti-mouse (1:10,000) or goat-anti rabbit (1:10,000) (Jackson ImmunoResearch). Detection of protein expression was assessed using the Western Lightning Plus-ECL detection Kit (PerkinElmer) and imaged with the ChemiDoc-Touch Imaging System (Bio-Rad).