Immunoblot Analysis of Fusion Proteins

Immunoblots were analyzed as described in Gardezi et al. (2016 (link)). Immunoblots were imaged with the ChemiDoc (Bio-Rad, Hercules, CA, USA) with a broad range of exposure times. For each experiment, protein band intensities were quantified by densitometry from a common blot at a single exposure selected for clear bands without saturation. Background counts were subtracted using an automated rolling disk subtraction. Protein band intensities were normalized to a single control condition for each experiment. For experiments comparing different fusion proteins, intensities were normalized to the C2 fusion protein intensity. For peptide experiments, intensities were normalize to the control peptide condition as described previously (Gardezi et al., 2016 (link)).
Fusion protein concentrations were visualized using Coomassie stain (Sigma-Aldrich) and imaged with the ChemiDoc (Bio-Rad) Coomassie stain protein gel function. Concentrations were quantified by densitometry and background was subtracted using an automated rolling disk subtraction. Fusion protein concentration was used as a loading control so that SV2 (I_SV2) and STG (I_STG) protein intensities were normalized to the fusion protein concentration (I_FP) from the same lane, hence %SV-PD was calculated as I_SV2/I_FP or I_STG/I_FP respectively.