Optimized semi-quantitative blot analysis in infection assays using the Stain-Free technology

•Validation of novel methods for the quantification of western blot signals•Disclosing the level of interference caused by bacterial proteins during semi-quantitative blot analysis•Adaptation of normalization procedures depends on bacteria and cell lines used.

Western blots are a commonly used method for protein detection and quantification in biological samples. Compensation of loading variations is achieved by housekeeping protein (HKP) normalization and/or total protein normalization (TPN). However, under infection conditions, HKP normalization, traditionally used in cell biology for quantification of western blots, can be problematic. Binding of microbes to target cells via specific receptors can induce signal transduction events resulting in drastic changes in the level of expression of HKPs. Additionally, samples collected after infection assays will include cellular and microbial proteins altering the analysis with TPN. Here we demonstrate under experimental infection conditions, how a reliable semi-quantitative analysis of proteins in western blots can be achieved using the Stain-Free technology.