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)