|H M R Mk||Endogenous||17||Rabbit|
Western blot analysis of extracts from HeLa, C6 and COS cells, untreated or treated with Trichostatin A (TSA) #9950 (400 nM for 18 h), using Acetyl-Histone H3 (Lys56) Antibody (upper) and Histone H3 Antibody #9715 (lower).Learn more about how we get our images.
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 datasheet or 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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
Acetyl-Histone H3 (Lys56) Antibody detects endogenous levels of histone H3 only when acetylated on Lys56. This antibody does not cross-react with histone H3 acetylated on lysines 9, 14, 18 or 27.
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of histone H3 in which Lys56 is acetylated. Antibodies are purified by protein A and peptide affinity chromatography.
Modulation of chromatin structure plays an important role in the regulation of transcription in eukaryotes. The nucleosome, made up of DNA wound around eight core histone proteins (two each of H2A, H2B, H3, and H4), is the primary building block of chromatin (1). The amino-terminal tails of core histones undergo various post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (2-5). These modifications occur in response to various stimuli and have a direct effect on the accessibility of chromatin to transcription factors and, therefore, gene expression (6). In most species, histone H2B is primarily acetylated at Lys5, 12, 15, and 20 (4,7). Histone H3 is primarily acetylated at Lys9, 14, 18, 23, 27, and 56. Acetylation of H3 at Lys9 appears to have a dominant role in histone deposition and chromatin assembly in some organisms (2,3). Phosphorylation at Ser10, Ser28, and Thr11 of histone H3 is tightly correlated with chromosome condensation during both mitosis and meiosis (8-10). Phosphorylation at Thr3 of histone H3 is highly conserved among many species and is catalyzed by the kinase haspin. Immunostaining with phospho-specific antibodies in mammalian cells reveals mitotic phosphorylation at Thr3 of H3 in prophase and its dephosphorylation during anaphase (11).
Acetylation of histone H3 on Lys56 is critical for proper packaging of DNA into chromatin during DNA replication and DNA damage repair (12-14). Histone H3 is acetylated on Lys56 by CBP and p300 in response to DNA damage induced by treatment of cells with γ radiation, ultraviolet light, MMS, or hydroxyurea (14). Following DNA damage, chromatin assembly factor 1 protein (CAF-1) incorporates acetylated histones into chromatin at sites of DNA repair (14). The class III histone deacetylases (HDACs) SirT1, SirT2 and SirT6 have been shown to deacetylate histone H3 at Lys56 (14,15); however, treatment of cells with sodium butyrate or trichostatin A also leads to increased acetylation, implicating a class I or class II HDAC as an additional histone H3 Lys56 deacetylase (14). Histone H3 Lys56 acetylation is high in multiple types of cancer, and acetylation levels directly correlate with cellular dedifferentiation and tumorigenicity (14).
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