ChIPAb+ Acetyl-Histone H3 (Lys18) - ChIP Validated Antibody and Primer Set

17 kDa

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 (Lys18) set includes the Acetyl-Histone H3 (Lys18) antibody, a negative control normal rabbit serum, and control primers which amplify a 166 bp region of human GAPDH promoter. The Acetyl-Histone H3 (Lys18) 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 (Lys18) -associated chromatin.

Histone H3 is one of the 5 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.

Acetylation of histone H3 occurs at several different lysine positions in the histone tail and is performed by a family of enzymes known as Histone Acetyl Transferases (HATs).

KLH-conjugated, synthetic peptide (GKAPRAcKQLASK-C) corresponding to amino acids 13-23 of yeast Histone H3 acetylated on lysine 18with a C-terminal cysteine added for conjugation purposes

Chromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared from HeLa cells (1 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µL of either Normal Rabbit Serum , or 2 µL of Anti-Acetyl-Histone H3 (Lys18)and the Magna ChIP^® A Kit (Cat. # 17-610). Successful immunoprecipitation of Acetyl-Histone H3 (Lys18) associated DNA fragments was verified by qPCR using Control Primers specific for the human GAPDH promoter region as a positive locus, and β-globin primers as a negative locus. 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.

ChIP-Sequencing:
Representative lot data.
Chromatin immunoprecipitation was performed using the Magna ChIP HiSens kit (cat# 17-10460), 2 µg of anti-acetyl-Histone H3 (Lys18) antibody (cat# 17-10111), 20 µL Protein A/G beads , and 1e6 crosslinked HeLa cell chromatin followed by DNA purification using magnetic beads. Libraries were prepared from Input and ChIP DNA samples using standard protocols with Illumina barcoded adapters, and analyzed on Illumina HiSeq instrument. An excess of ten million reads from FastQ files were mapped using Bowtie (http://bowtie-bio.sourceforge.net/manual.shtml) following TagDust (http://genome.gsc.riken.jp/osc/english/dataresource/) tag removal. Peaks were identified using MACS (http://luelab.dfci.harvard.edu/MACS/), with peaks and reads visualized as a custom track in UCSC Genome Browser (http://genome.ucsc.edu) from BigWig and BED files.

Western Blot Analysis:
Representative lot data.
Recombinant histone H3 (lane 1, Catalog # 14-494) and acid extracts from sodium butyrate treated (lane 2) and untreated (lane 3) HeLa cells (Catalog # 17-305) were probed with anti acetyl- Histone H3 (Lys18) (1:10,000 dilution).
Arrow indicates acetyl histone H3 (Lys18) (17 kDa).

Dot Blot:
Representative lot data.
40 ng and 4ng amounts of histone peptides with various modifications (see table 1) were transferred to PVDF membrane and probed with Anti-Acetyl-Histone H3 (Lys18) antibody (1:5000 dilution). Proteins were visualized using a goat anti-rabbit IgG conjugated to HRP and a chemiluminescence detection system. Image from a 60 second exposure is shown.

Research Category
Epigenetics & Nuclear Function

Research Sub Category
Histones

This ChIPAb+ Acetyl-Histone H3 (Lys18) -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.

Recognizes Histone H3 acetylated on lysine 18.

25 assays per set. Recommended use: ~2 μL of antibody per chromatin immunoprecipitation (dependent upon biological context).

Anti-Acetyl-Histone H3 (Lys18) (rabbit polyclonal). One vial of 50 µL of rabbit serum containing 0.05% sodium azide and 30% glycerol. Store at -20°C.
Normal Rabbit Serum. One vial of 100 μL of antiserum containing 0.05% sodium azide. Store at -20°C.
Control Primers, human GAPDH promoter. One vial containing 75 μL of 5 μM each of primer specific for human GAPDH promoter. Store at -20°C.
FOR: TAC TAG CGG TTT TAC GGG CG
REV: TCG AAC AGG AGG AGC AGA GAG CGA

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.

Chromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared from HeLa cells (1 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µL of either Normal Rabbit Serum, or 2 µL of Anti-Acetyl-Histone H3 (Lys18)and the Magna ChIP^® A Kit (Cat. # 17-610). Successful immunoprecipitation of Acetyl-Histone H3 (Lys18) associated DNA fragments was verified by qPCR using Control Primers specific for the human GAPDH promoter region (Figure 1).
Please refer to the EZ-Magna ChIP A (Cat. # 17-408) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.

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

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

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

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.