Cell Signaling Technology

Product Pathways - Chromatin Regulation / Epigenetics

Acetyl-Histone H4 (Lys5) (D12B3) Rabbit mAb #8647

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IHC-P IF-IC ChIP H M R Mk (C) (Dm) (X) (Z) (B) (Pg) (Hr) (Ce) Endogenous 11 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  C=Chicken  Dm=D. melanogaster  X=Xenopus  Z=Zebrafish  B=Bovine  Pg=Pig  Hr=Horse  Ce=C. elegans
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Acetyl-Histone H4 (Lys5) (D12B3) Rabbit mAb recognizes endogenous levels of histone H4 protein only when acetylated at Lys5. This antibody does not cross-react with histone H4 acetylated at Lys8, Lys12, or Lys16.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding acetylated Lys5 of human histone H4 protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Acetyl-Histone H4 (Lys5) (D12B3) Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa and C2C12 cells, untreated (-) or treated with Trichostatin A (TSA) #9950 (1 μM, 18 hr; +), using Acetyl-Histone H4 (Lys5) (D12B3) Rabbit mAb (upper) or Histone H4 (L64C1) Mouse mAb #2935 (lower).

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast using Acetyl-Histone H4 (Lys5) (D12B3) Rabbit mAb in the presence of non-acetyl-histone H4 (Lys5) peptide (left) or acetyl-histone H4 (Lys5) peptide (right).


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Acetyl-Histone H4 (Lys5) (D12B3) Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Acetyl-Histone H4 (Lys5) (D12B3) Rabbit mAb.

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Trichostatin A (TSA) #9950 (right) using Acetyl-Histone H4 (Lys5) (D12B3) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).


Chromatin IP

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) (D12B3) Rabbit mAb 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, SimpleChIP® Human MyoD1 Exon 1 Primers #4490, 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.

ELISA-Peptide

ELISA-Peptide

Acetyl-Histone H4 (Lys5) (D12B3) Rabbit mAb specificity was determined by peptide ELISA. The graph depicts the binding of the antibody to pre-coated acetyl-histone H4 (Lys5) peptide in the presence of increasing concentrations of various competitor peptides. As shown, only the acetyl-histone H4 (Lys5) peptide competed away binding of the 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, 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).

  1. Peterson, C.L. and Laniel, M.A. (2004) Curr Biol 14, R546-51.
  2. Jaskelioff, M. and Peterson, C.L. (2003) Nat Cell Biol 5, 395-9.
  3. Roth, S.Y. et al. (2001) Annu Rev Biochem 70, 81-120.
  4. Workman, J.L. and Kingston, R.E. (1998) Annu Rev Biochem 67, 545-79.
  5. Hansen, J.C. et al. (1998) Biochemistry 37, 17637-41.
  6. Yang, X.J. (2004) Bioessays 26, 1076-87.
  7. Haberland, M. et al. (2009) Nat Rev Genet 10, 32-42.
  8. Haigis, M.C. and Sinclair, D.A. (2010) Annu Rev Pathol 5, 253-95.

Application References

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