6.1. Western-blot analysis
As a first step in these analyses, a detection and semi-quantification of the targeted acetylated protein can be made by western blot analysis. For this reason, is necessary to extract the proteins from the cells or tissues using lysis buffers with chaotropic agents and detergents; in addition, to mechanical cell disruption methods such as heating, bead beating, ultrasonication, or mechanical agitation, to facilitate the protein extractions. Then, proteins can be purified and separated by gel electrophoresis for the subsequent transfer to nitrocellulose or polyvinylidene difluoride (PVDF) membranes to finally be detected using specific antibodies against acetylated proteins (Diallo et al., 2019; Yu et al., 2008; Hirano, 2012).
In western blot analysis, two types of antibodies can be used: (1) site-specific antibodies for recognition of a consensus amino acid sequences containing acetyl-lysine residue(s); (2) pan-specific antibodies for recognition of acetyl-lysine residues regardless of the surrounding amino acid sequence (Diallo et al., 2019; Komatsu et al., 2005).
The study of Schilling and coworkers on the effect of different carbon sources on protein acetylation in E. coli through western blot analysis showed that the global acetylation profile increased steadily independent of the carbon source used for their growth, with a higher number of acetylated proteins after the cells enter the stationary phase. Furthermore, immunoblot analysis revealed a higher number of acetylated proteins when the medium was supplemented with the carbon source after an incubation period. With these results, the authors suggest that carbon-induced protein acetylation in the stationary phase only occurs if cells are exposed to a supplemental carbon source in the stationary phase (Schilling et al., 2015). However, this approach usually presents a low sensitive and non-specific signal, the needs to introduce negative controls to ensure the anti-acetyl lysine antibody specificity, and it is not possible to know the number/assignment of specific acetylation sites and protein identification (Diallo et al., 2019)