WB, IF-IC, FC-FP, ChIP, ChIP-seq
H M R Mk
Endogenous
23
Rabbit IgG
#P33778
3018
Product Information
Product Usage Information
For optimal ChIP and ChIP-seq results, use 2.5 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.
| Application | Dilution |
|---|---|
| Western Blotting | 1:1000 |
| Immunofluorescence (Immunocytochemistry) | 1:800 - 1:3200 |
| Flow Cytometry (Fixed/Permeabilized) | 1:400 - 1:1600 |
| Chromatin IP | 1:200 |
| Chromatin IP-seq | 1:200 |
Storage
Specificity / Sensitivity
Species Reactivity:
Human, Mouse, Rat, Monkey
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the carboxy terminus of the human histone H2B protein in which Lys120 is mono-ubiquitylated.
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 ubiquitylation (1). Ubiquitin is a conserved 76 amino acid peptide unit that can be covalently linked to many cellular proteins by the ubiquitylation process. Three components are involved in this protein-ubiquitin conjugation process. Ubiquitin is first activated by forming a thiolester complex with the activation component E1; the activated ubiquitin is subsequently transferred to the ubiquitin-carrier protein E2, then from E2 to ubiquitin ligase E3 for final delivery to the epsilon-NH2 of the target protein lysine residue (2). Histone H2B is mono-ubiquitylated on lysine 120 during transcriptional activation by the RAD6 E2 protein in conjunction with the BRE1A/BRE1B E3 ligase (also known as RNF20/RNF40) (3). The RAD6/BRE1 complex is recruited to gene promoters during activation by the PAF complex, an RNA polymerase II-associated protein complex that regulates transcriptional elongation (3-5). Mono-ubiquitylated histone H2B lysine 120 is associated with the transcribed region of active genes (3,6). Mono-ubiquitylation of histone H2B stimulates transcriptional elongation by facilitating FACT-dependent chromatin remodeling (7,8). In addition, it is essential for subsequent methylation of histone H3 lysines 4 and 79, two additional histone modifications that regulate transcriptional initiation and elongation (9). Interestingly, de-ubiquitylation of histone H2B lysine 120 by USP22, a subunit of the human SAGA histone acetyltransferase complex, is a required step in transcriptional activation (10). Thus, it appears that the ubiquitylation state of histone H2B is dynamic during transcription and may serve as an intermediate step in transcriptional activation.
- Peterson, C.L. and Laniel, M.A. (2004) Curr Biol 14, R546-51.
- Liu, F. and Walters, K.J. (2010) Trends Biochem Sci 35, 352-60.
- Kim, J. et al. (2009) Cell 137, 459-71.
- Wood, A. et al. (2003) J Biol Chem 278, 34739-42.
- Xiao, T. et al. (2005) Mol Cell Biol 25, 637-51.
- Minsky, N. et al. (2008) Nat Cell Biol 10, 483-8.
- Pavri, R. et al. (2006) Cell 125, 703-17.
- Fleming, A.B. et al. (2008) Mol Cell 31, 57-66.
- Shilatifard, A. (2006) Annu Rev Biochem 75, 243-69.
- Wyce, A. et al. (2004) Novartis Found Symp 259, 63-73; discussion 73-7, 163-9.
Species Reactivity
Species reactivity is determined by testing in at least one approved application (e.g., western blot).
Western Blot Buffer
IMPORTANT: 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.
Applications Key
WB: Western Blotting IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq
Cross-Reactivity Key
H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected
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