An immunoblot based assay to quantitate free and total ubiquitin content of whole protein extracts.
In this blot, parallel pupal samples (from stage P4 pupae) homogenized in buffers F (lanes 1 and 2; +NEM) and buffer T (lanes 3 and 4; -NEM) were loaded and polyclonal anti-Ub antibody at a dilution of 1:1000 was used to detect ubiquitin. Only the intensity of the free monoubiquitin band (just below the 10 kDa mark) was determined and used for quantification. 5 μg total protein extracts were loaded to lanes 1 and 2, while samples were diluted twofold before loading to lanes 3 and 4 to avoid overloading the monoubiquitin band. Ub standards of 0.5, 1, 2 and 3 pmol were loaded to lane 5–8 respectively, for the calibration curve. The inset shows the calibration curve that was used to calculate Ub concentrations in these samples. Band intensities were plotted against Ub standards and a regression line equation was generated by applying the four parameter curve fit model (R2 = 0.9978).
