Western blot analysis of lysates from HeLa and C6 cells, untreated (-) or treated with TSA #9950 (1 μM, 18 hr; +), using Acetyl-Histone H3 (Lys23) (D6Y7M) Rabbit mAb (upper) and Histone H3 (D1H2) XP® Rabbit mAb #4499 (lower).
Acetyl-Histone H3 (Lys23) (D6Y7M) Rabbit mAb antibody specificity was determined by western blotting. HeLa cell lysates, untreated (-) or treated with TSA #9950 (1 μM, 18 hr; +), were probed with Acetyl-Histone H3 (Lys23) (D6Y7M) Rabbit mAb (panel A) or Acetyl-Histone H3 (Lys23) (D6Y7M) Rabbit mAb pre-adsorbed with 1.5 μM of various competitor peptides (panels B-K). As shown, only the acetyl-histone H3 (Lys23) peptide competed away binding of the antibody.
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Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
Acetyl-Histone H3 (Lys23) (D6Y7M) Rabbit mAb recognizes endogenous levels of histone H3 protein only when acetylated at Lys23. This antibody does not cross-react with histone H3 acetyl-lysine 9, 14, 18, 27, or 56.
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding acetyl-Lys23 of human histone H3 protein.
The nucleosome, made up of four core histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. Originally thought to function as a static scaffold for DNA packaging, histones have now been shown to be dynamic proteins, undergoing multiple types of post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (1,2). Histone acetylation occurs mainly on the amino-terminal tail domains of histones H2A (Lys5), H2B (Lys5, 12, 15, and 20), H3 (Lys9, 14, 18, 23, 27, 36 and 56), and H4 (Lys5, 8, 12, and 16) and is important for the regulation of histone deposition, transcriptional activation, DNA replication, recombination, and DNA repair (1-3). Hyper-acetylation of the histone tails neutralizes the positive charge of these domains and is believed to weaken histone-DNA and nucleosome-nucleosome interactions, thereby destabilizing chromatin structure and increasing the accessibility of DNA to various DNA-binding proteins (4,5). In addition, acetylation of specific lysine residues creates docking sites for a protein module called the bromodomain, which binds to acetylated lysine residues (6). Many transcription and chromatin regulatory proteins contain bromodomains and may be recruited to gene promoters, in part, through binding of acetylated histone tails. Histone acetylation is mediated by histone acetyltransferases (HATs), such as CBP/p300, GCN5L2, PCAF, and Tip60, which are recruited to genes by DNA-bound protein factors to facilitate transcriptional activation (3). Deacetylation, which is mediated by histone deacetylases (HDAC and sirtuin proteins), reverses the effects of acetylation and generally facilitates transcriptional repression (7,8).
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