Immunofluorescence analysis of Histone H3 [AcK9] was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton™ X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with ABfinity™ Histone H3 [AcK9] Recombinant Rabbit Monoclonal Antibody (701269) at 1 µg-2 µg in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour® 488 Goat Anti-Rabbit IgG Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 Phalloidin (A12381). Panel d is a merged image showing nuclear localization of Histone H3 [AcK9]. Panel e shows competition with Histone H3 [AcK9] peptide. The images were captured at 20X magnification.
|Tested species reactivity||Fruit fly, Human, Rat|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Acetylated peptide corresponding to amino acids 6–12 of human Histone H3 [AcK9]|
|Contains||0.09% sodium azide|
|Storage Conditions||Maintain refrigerated at 2-8°C for up to 1 month. For long term storage store at -20°C|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||3µg|
|Flow Cytometry (Flow)||1-3µg/10^6 cells|
|Western Blot (WB)||1:1500|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
701269 was successfully used to detect H3K9ac in WB, IF, IHC, ChIP and flow applications.
This antibody is predicted to react with mouse, rat, non-human primate and rabbit based on sequence homology.
ABfinity™ recombinant antibodies are rabbit monoclonal antibodies, unmatched for producing superior results. ABfinity™ antibodies are developed by immunizing animals, screening for functionality, cloning the immunogen-specific antibody genes into high-level mammalian expression vectors, produced on a large scale, and purified with Protein A.
ABfinity™ monoclonal antibodies resemble rabbit monoclonals isolated from serum or produced by hybridomas, but demonstrate greater specificity and sensitivity. Because ABfinity™ recombinant antibodies are derived from cloned DNA sequences of the heavy and light antibody chains, they are not susceptible to cell-line drift or lot-to-lot variation, thus allowing for peak specificity and performance.
Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.
Histone H3 is one of the four core proteins of the nucleosome, and it is involved in transcription regulation, DNA repair, DNA replication and chromosomal stability. The N-terminal tail of Histone H3 undergoes many post-translational modifications, including phosphorylation, acetylation, multiple methylation, ubiquitination, and ADP-ribosylation to achieve its diverse functions. HistoneH3 is acetylated and deacetylated on N-terminal lysine residues. Acetylation removes the positive charge on the histone, decreasing the interaction with the negatively charged phosphate groups of DNA, and resulting in a more relaxed structure associated with greater levels of gene transcription. Acetylation of histone H3 at lysine 9 (H3K9Ac) is one of the most well-known epigenetic markers enriched in the promoter region of activated genes.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.