Quantitative Protein Measurement in Tissue Samples

Fresh frozen slice culture tissues were homogenized in NP-40 lysis buffer supplemented with protease and phosphatase inhibitors [36 (link)]. Protein concentrations were determined with the bi cinchoninic acid (BCA) assay (Pierce, Rockford, IL). Immunoreactivity was measured in 4 replicate 100 ng protein samples by direct binding ELISA [36 (link)], and protein loading was subsequently quantified by measuring immune reactivity to large acidic ribosomal protein (RPLPO) [36 (link)]. Primary antibodies were diluted to 0.1-0.5 pg/ml, and their binding was detected with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000; Pierce, Rockford, IL) and Amplex Ultra Red soluble fluorophore (Molecular Probes, Eugene, OR). Amplex Red fluorescence fluorescent light units (FLU) were measured in a SpectraMax M5 (Ex 530/Em 590). Subsequently, the samples were incubated with biotin-conjugated polyclonal antibodies to RPLPO, and immunoreactivity was detected with streptavidin-conjugated alkaline phosphatase (1:1000; Vector, Burlingame, CA) and the 4-Methylumbelliferyl phosphate (4-MUP) fluorophore (Molecular Probes, Eugene, OR) (Ex360/Em450). Binding specificity was assessed with negative control incubations in which the primary or secondary antibody was omitted. The calculated ratios of specific protein/RPLPO fluorescence were used for inter-group statistical comparisons.