Chromatin Regulation / Acetylation

Acetyl-Histone H4 (Lys5) Antibody #9672

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Applications Key:   W = Western Blotting  IP = Immunoprecipitation  IHC-P = Immunohistochemistry (Paraffin)  IF-IC = Immunofluorescence (Immunocytochemistry)  ChIP = Chromatin IP
Reactivity Key: B = Bovine  C = Chicken  Dm = D. melanogaster  H = Human  Hr = Horse  M = Mouse  Mk = Monkey  Pg = Pig  R = Rat  X = Xenopus  Z = Zebrafish  
Species cross-reactivity is determined by Western blot.

Protocols

9672

Western Blotting, ChIP Agarose, ChIP Magnetic, IHC / Paraffin, Immunoprecipitation,

Specificity / Sensitivity

Acetyl-Histone H4 (Lys5) Antibody detects endogenous levels of histone H4 only when acetylated on Lys5. This antibody does not cross-react with histone H4 acetylated on lysines 8, 12, or 16.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of histone H4 in which Lys5 is acetylated. Antibodies are purified by protein A and peptide affinity chromatography.

IF-IC

Immunofluorescent analysis of HeLa cells, untreated (left) or treated with Trichostatin A (TSA) #9950 (right), using Acetyl-Histone H4 (Lys5) Antibody (green) and Golgin-97 Antibody (red). Actin filaments were labeled with a dye conjugated phalloidin (pseudocolored blue).

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Acetyl-Histone H4 (Lys5) Antibody.

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Acetyl-Histone H4 (Lys5) Antibody.

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 20 μl of Acetyl-Histone H4 (Lys5) Antibody or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP™ Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP™ Human GAPDH Exon 1 Primers #5516, SimpleChIP™ Human RPL30 Exon 3 Primers #7014, and SimpleChIP™ Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Western Blotting

Western blot analysis of extracts from various cell lines using Acetyl-Histone H4 (Lys5) Antibody.

Background

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, 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 access 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). De-acetylation, which is mediated by histone deacetylases (HDAC and Sirtuin proteins), reverses the effects of acetylation and generally facilitates transcriptional repression (7,8).

1. Peterson, C.L. and Laniel, M.A. et al. (2004) Curr Biol14, R546 - 51.
2. Jaskelioff, M. and Peterson, C.L. et al. (2003) Nat Cell Biol5, 395 - 9.
3. Roth, S.Y. et al. (2001) Annu Rev Biochem70, 81 - 120.
4. Workman, J.L. and Kingston, R.E. et al. (1998) Annu Rev Biochem67, 545 - 79.
5. Hansen, J.C. et al. (1998) Biochemistry37, 17637 - 41.
6. Yang, X.J. et al. (2004) Bioessays26, 1076 - 87.
7. Haberland, M. et al. (2009) Nat Rev Genet10, 32 - 42.
8. Haigis, M.C. and Sinclair, D.A. et al. (2010) Annu Rev Pathol5, 253 - 95.

Application References

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Companion Products

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• 7720 Prestained Protein Marker, Broad Range (Premixed Format)
• 7727 Biotinylated Protein Ladder Detection Pack
• 2960 Histone H4 (L64C1) Mouse mAb (ChIP Formulated)
• 7003 20X LumiGLO® Reagent and 20X Peroxide
• 9002 SimpleChIP™ Enzymatic Chromatin IP Kit (Agarose Beads)
• 8112 SignalStain® Antibody Diluent
• 7074 Anti-rabbit IgG, HRP-linked Antibody
• 8114 SignalStain® Boost IHC Detection Reagent (HRP, Rabbit)

This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.