ab61257 was affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific acetylated peptide. The antibody against non-acetylated peptide was removed by chromatography using non-acetylated peptide corresponding to the acetylation site.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/50 - 1/100.
1/500 - 1/1000. Detects a band of approximately 85 kDa.
Use a concentration of 1 µg/ml.
In the nucleus, DNA is tightly packed into nucleosomes generating an environment which is highly repressive towards DNA processes such as transcription. Acetylation of lysine residues within proteins has emerged as an important mechanism used by cells to overcome this repression. The acetylation of non-histone proteins such as transcription factors, as well as histones appears to be involved in this process. Acetylation may result in structural transitions as well as specific signaling within discrete chromatin domains. The role of acetylation in intracellular signaling has been inferred from the binding of acetylated peptides by the conserved bromodomain. Furthermore, recent findings suggest that bromodomain/acetylated-lysine recognition can serve as a regulatory mechanism in protein-protein interactions in numerous cellular processes such as chromatin remodeling and transcriptional activation.
The reversible lysine acetylation of histones and non-histone proteins plays a vital role in the regulation of many cellular processes including chromatin dynamics and transcription, gene silencing, cell cycle progression, apoptosis, differentiation, DNA replication, DNA repair, nuclear import, and neuronal repression. More than 20 acetyltransferases and 18 deacetylases have been identified so far, but the mechanistic details of substrate selection and site specificity of these enzymes remain unclear. Over 40 transcription factors and 30 other nuclear, cytoplasmic, bacterial, and viral proteins have been shown to be acetylated in vivo.
ICC/IF image of ab61257 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab61257 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Western blot - Anti-acetyl Lysine antibody (ab61257)Image courtesy of an anonymous Abreview.
293 histone (acid extracted) loaded at 2µg. Blocking step performed using 0.3% BSA for 12 hours at 4°C.ab61257 used at a 1/1000 dilution for 1 hour at 25°C. The secondary used was an HRP conjugated donkey anti-rabbit IgG polyclonal used at a 1/2000 dilution.
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