Quantitation of Renilla Luciferase Protein Expression

HEK293T cells were seeded in 6-well plates (5 × 10⁵ cells/well) and the next day were transfected with 5 μg of Renilla luciferase (RLuc)-expression constructs, using the calcium phosphate method as described in [35 (link)]. At 48 h post transfection, cells were washed with ice-cold PBS and lysed in 250 μL of cracking buffer (0.09 M Tris·Cl, pH 6.8. 20% glycerol, 2% SDS, 0.02% bromophenol blue, 0.1 M DTT). Ten microliters of each sample was analyzed by SDS page/Western blotting as described previously [26 (link)], using the α-RLuc mouse monoclonal antibody (Ab; MAB4400, Merck Millipore, Burlington, MA, USA; 1:4000) and the goat α-mouse immunoglobulin Ab conjugated to horseradish peroxidase (sc-2055, Santa Cruz Biotech, Dallas, TX, USA; 1:10,000), both diluted in PBS/BSA 3% (w/v). Immunoblots were developed with the ECL prime substrate (Amersham, Little Chalfont, UK) in combination with Carestream^® Kodak^® BioMax^® light autoradiography films (Merck Millipore).