ChIPAb+ Trimethyl-Histone H3 (Lys79) - ChIP Validated Antibody and Primer Set

General description

All ChIPAb+ antibodies are individually validated for chromatin precipitation, every lot, every time. Each ChIPAb+ antibody set includes control primers (tested every lot by qPCR) to biologically validate your IP results in a locus-specific context. The qPCR protocol and primer sequences are provided, allowing researchers to validate ChIP protocols when using our antibody in their chromatin context. Each set also includes a negative control antibody to ensure specificity of the ChIP reaction.
The ChIPAb+ Trimethyl-Histone H3 (Lys79) set includes the Trimethyl-Histone H3 (Lys79) antibody, a negative control normal rabbit IgG, and qPCR primers which amplify a 166 bp region of human GAPDH. The Trimethyl-Histone H3 (Lys79) and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of Trimethyl-Histone H3 (Lys79)-associated chromatin.

Histones are highly conserved proteins that serve as the structural scaffold for the organization of nuclear DNA into chromatin. The four core histones, H2A, H2B, H3, and H4, assemble into an octamer (2 molecules of each). Subsequently, 146 base pairs of DNA are wrapped around the octamer, forming a nucleosome, the basic subunit of chromatin. Histone modifications regulate DNA transcription, repair, recombination, and replication. The most commonly studied modifications are acetylation, phosphorylation, methylation, and ubiquitination. Lysine methylation occurs in three distinct states, having either one (me1), two (me2) or three (me3) methyl groups attached to the amine group of the lysine side chain. Methylation of Lysine 79 in Histone 3 acts as a marker of inactive chromatin regions. Methylation by Dot1 methylase on lysine-79 (Lys79) of Histone H3 is criticial for transcriptional silencing, and it is thought that silencing proteins such as Sir3 function by blocking Dot1 methylation.

Specificity

Broad species reactivity is expected.

This antibody recognizes Histone H3 Trimethylated on Lys79.

Immunogen

Epitope: Trimethyl Lys79

Linear Peptide corresponding to human Histone H3 methylated at Lys79.

Application

Chromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared from HeLa cells (1 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using either 1 µg of normal rabbit IgG, or 1 µg of Anti-trimethyl-Histone H3 (Lys79) and the Magna ChIP^® A Kit (Cat. # 17-610).
Successful immunoprecipitation of trimethyl-Histone H3 (Lys79) associated DNA fragments was verified by qPCR using Control Primers as a positive locus, and human β-globin as a negative locus (Figure 2). Data is presented as percent input of each IP sample relative to input chromatin for each amplicon and ChIP sample as indicated.
Please refer to the EZ-Magna ChIP A (Cat. # 17-408) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.
Western Blot Analysis:
Representative lot data.
HeLa acid extract (lane 1) and recombinant Histone H3 (Cat # 14-494) (lane2) lysates were resolved by electrophoresis, transferred to PVDF membrane and probed with Anti-trimethyl Histone H3 (Lys79) (0.1 μg/mL). Proteins were visualized using a Donkey Anti-rabbit IgG conjugated to HRP and a chemiluminescence detection system.
Arrow indicates Histone H3 (~17 kDa) (Figure 3).
Multiplex Immunoassay: Representative lot data.
Specificity of interaction of anti-trimethyl histone H3 Lys79 antibody was assayed using the Luminex multiplex immunoassay platform. Anti-trimethyl-Histone H3 (Lys79) diluted to 0.01 µg/mL concentration was incubated with a cocktail of xMAP microspheres conjugated to the following histone H3 peptides, monomethyl Lys79, unmodified Lys79, trimethyl Lys 27, trimethyl Lys 9, trimethyl Lys79 processed and detected using standard xMAP protocols (Figure 4).

Research Category
Epigenetics & Nuclear Function

Research Sub Category
Histones

Chromatin Biology

This ChIPAb+ Trimethyl-Histone H3 (Lys79) -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.

Packaging

25 assays per set. Recommended use: ~1 μg of antibody per chromatin immunoprecipitation (dependent upon biological context).

Quality

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using either 1 µg of normal rabbit IgG, or 1 µg of Anti-trimethyl-Histone H3 (Lys79) and the Magna ChIP^® A Kit (Cat. # 17-610). Successful immunoprecipitation of trimethyl-Histone H3 (Lys79) associated DNA fragments was verified by qPCR using Control Primers, (Figure 1).
Please refer to the EZ-Magna ChIP A (Cat. # 17-408) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.

Target description

~ 17 kDa

Physical form

Affinity purified

Anti-Trimethyl-Histone H3 (Lys79) (rabbit polyclonal), Part No. CS204342. One vial containing 25 µg of purified rabbit polyclonal in buffer containing 0.1 M Tris-Glycine (pH 7.4, 150 mM NaCl) with 0.05% sodium azide before the addition of 30% glycerol.
Store at -20°C.
Normal Rabbit IgG, Part No. PP64B. One vial containing 125 µg Rabbit IgG in 125 µL storage buffer containing 0.05% sodium azide.
Store at -20°C.
Control Primers, Part No. 22-004. One vial containing 75 μL of 5 μM of each primer specific for human GAPDH. Store at -20°C.
FOR: TAC TAG CGG TTT TAC GGG CG
REV: TCG AAC AGG AGG AGC AGA GAG CGA

Storage and Stability

Stable for 1 year at -20°C from date of receipt. Handling Recommendations: Upon first thaw, and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance. Note: Variability in freezer temperatures below -20°C may cause glycerol containing solutions to become frozen during storage.

Analysis Note

Control
Includes negative control normal rabbit IgG and primers specific for human GAPDH.

Other Notes

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

Legal Information

MAGNA CHIP is a registered trademark of Merck KGaA, Darmstadt, Germany

UPSTATE is a registered trademark of Merck KGaA, Darmstadt, Germany

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.