ChIPAb+ Acetyl-Histone H3 (Lys4) - 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+ Acetyl-Histone H3 (Lys4) set includes the Acetyl-Histone H3 (Lys4) antibody, a negative control antibody (normal rabbit IgG), and qPCR primers which amplify a 166 bp region of human GAPDH. The Acetyl-Histone H3 (Lys4) and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of Acetyl-Histone H3 (Lys4)-associated chromatin.

Histone H3 is one of the five main histone proteins involved in the structure of chromatin in eukaryotic cells. Featuring a main globular domain and a long N-terminal tail, H3 is involved with the structure of the nucleosomes of the ′beads on a string′ structure. Histone proteins are highly post-translationally modified, and Histone H3 is the most extensively modified of the five histones. The term "Histone H3" alone is purposely ambiguous in that it does not distinguish between sequence variants or modification state. Histone H3 is an important protein in the emerging field of epigenetics, where its sequence variants and variable modification states are thought to play a role in the dynamic and long term regulation of genes.

Specificity

Recognizes Histone H3 acetylated on Lys4.

Wide range of cross-reactivity expected based on sequence homology.

Immunogen

Epitope: Acetylated Lys4

KLH-conjugated, synthetic peptide containing the sequence …RTAcKQ… in which AcK corresponds to acetyl lysine 4 of human histone H3.

Application

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1 X 10⁶ cell equivalents per IP) were subjected to chromatin immunoprecipitation using 4 µL of Normal rabbit IgG or 4 µL Anti-Acetyl-Histone H3 (Lys4) and the Magna ChIP^® A Kit (Cat. # 17-610).
Successful immunoprecipitation of Acetyl-Histone H3 (Lys4) associated DNA fragments was verified by qPCR using Control Primers as a positive locus on the GAPDH promoter, and β-Globin primers as a negative locus. (Please see figures).
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.
Recombinant histone H3 (Catalog # 14-411, lane 1) and acid extracts from sodium butyrate treated (lane 2) and untreated (lane 3) HeLa cells (Catalog # 17-305) were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-acetyl-Histone H3 (Lys4) (1:1,000 dilution).
Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system. (Please see pictures).

Histone-Peptide Specificity Assay: Representative lot data.
A 1:1,000 – 1:5,000 dilution of anti-acetyl Histone H3 (Lys4) was incubated with a cocktail of microspheres conjugated to histone H3
peptides with the following modifications:

1. Unmodified, containing Lys 4, 9, 14 and 18
2. Acetyl-lysine 4
3. Acetyl-lysine 9
4. Acetyl-lysine 14
5. Acetyl-lysine 18
6. Acetyl-lysine 23
7. Acetyl-lysine 27

Unbound antibody was then removed by filtration. Peptide-antibody complexes were incubated with a biotin-conjugated anti-rabbit secondary antibody followed by incubation with a phycoerythrin-streptavidin conjugate. Fluorescence was read on a Luminex 100 instrument.
Median Fluorescence Intensity (MFI) is plotted (Please see figures).

Research Category
Epigenetics & Nuclear Function

Research Sub Category
Histones

This ChIPAb+ Acetyl-Histone H3 (Lys4) -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.

Packaging

25 assays per set. Recommended use: ~4 μL of antibody per chromatin immunoprecipitation (dependent upon biological context).

Components

Anti-Acetyl-Histone H3 (Lys4) (rabbit polyclonal)

Normal Rabbit IgG

Control Primers

Quality

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1 X 10⁶ cell equivalents per IP) were subjected to chromatin immunoprecipitation using 4 µL of Normal Rabbit IgG or 4 µL Anti-Acetyl-Histone H3 (Lys4) and the Magna ChIP^® A Kit (Cat. # 17-610). Successful immunoprecipitation of Acetyl-Histone H3 (Lys4) associated DNA fragments was verified by qPCR using Control Primers (Please see figures).

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-Acetyl-Histone H3 (Lys4) (rabbit polyclonal). One vial containing 100 µL of immunoaffinity purified rabbit polyclonal in storage buffer containing 0.05% sodium azide with 30% glycerol. Store at -20°C.

Normal Rabbit IgG. One vial containing 125 µg Rabbit IgG in 125 µL storage buffer containing 0.05% sodium azide. Store at -20°C.

Control Primers. One vial containing 75 μL of 5 μM 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 antibody and primers specific for human GAPDH.

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.