ChIPAb+ Acetyl-Histone H4 (Lys5) - ChIP Validated Antibody and Primer Set, rabbit monoclonal

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 H4 (Lys5) set includes the Acetyl-Histone H4 (Lys5) antibody, a negative control supernatant, and qPCR primers which amplify a 166 bp region of human GAPDH. The Acetyl-Histone H4 (Lys5) and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of Acetyl-Histone H4 (Lys5)-associated chromatin.

Histone H4 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 H4 is involved with the structure of the nucleosomes of the ′beads on a string′ structure. Acetylation of histone H4 occurs at several different lysine positions in the histone tail and is performed by a family of enzymes known as Histone Acetyl Transferases (HATs).

Specificity

Recognizes Histone H4 acetylated on Lys5.

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

Immunogen

Epitope: Acetylated Lys5

Peptide corresponding to Histone H4 containing the sequence [GRG-AcK-GGK] on which Lys5 is acetylated.

Application

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from untreated HeLa cells (1 X 10⁶ cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µL of either a negative control supernatant , or 2 µL of Anti-Acetyl-Histone H4 (Lys5) and the Magna ChIP^® A Kit (Cat. # 17-610).
Successful immunoprecipitation of acetyl-Histone H4 (Lys5) associated DNA fragments was verified by qPCR using control Primers flanking the human GAPDH promoter, or primers amplifying the promoter of human β-globin, which is transcriptionally inactive in HeLa cells (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.
Lysates from HeLa cells untreated or sodium butyrate treated (Lanes 1 and 2 respectively) were probed with anti-acetyl-Histone H4 (Lys5) (1:1,000 dilution).
Proteins were visualized using goat anti-rabbit. (Please see figures).

Western Blot Analysis/Peptide Inhibition: Representative lot data.
Sodium butyrate treated HeLa acid extract was resolved by electrophoresis, transferred to PVDF membrane and probed with Anti-Acetyl-Histone H4 (Lys5) (1:1000 dilution).
To demonstrate specificity, serum was preincubated with modified Histone H4 peptides:

Lane 1: No peptide
Lane 2: Acetyl-Histone H4 (K5) (Cat. # 12-343)
Lane 3: Acetyl-Histone H4 (K8) (Cat. # 12-344)
Lane 4: Acetyl-Histone H4 (K12) (Cat. # 12-345)
Lane 5: Acetyl-Histone H4 (K16) (Cat. # 12-346)
Lane 6: Acetyl-Histone H4 (K5, 8, 12, 16) (Cat. # 12-353)
Lane 7: Histone H4 (Cat. # 12-347)

Proteins were visualized using a donkey anti-rabbit IgG conjugated to HRP and visualized using a chemiluminescence detection system. (Please see figures).

Research Category
Epigenetics & Nuclear Function

Research Sub Category
Histones

This ChIPAb+ Acetyl-Histone H4 (Lys5) -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.

Packaging

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

Quality

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from untreated HeLa cells (1 X 10⁶ cell equivalents per IP) was subjected to chromatin immunoprecipitation using 2 µL of either negative control supernatant or 2 µL of Anti-Acetyl-Histone H4 (Lys5) and the Magna ChIP^® A Kit (Cat. # 17-610).
Successful immunoprecipitation of acetyl-Histone H4 (Lys5) associated DNA fragments was verified by qPCR using control ChIP Primers flanking the human GAPDH promoter (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

~11 kDa

Physical form

Anti-Acetyl-Histone H4 (Lys5) (rabbit monoclonal IgG). One vial containing 50 μL of rabbit monoclonal IgG cell culture supernatant in 0.1% sodium azide. Store at -20°C.

Negative Control Supernatant (rabbit). One vial containing 50 μL of cultured supernatant in 0.05% sodium azide. Store at -20°C.

Control Primers. One vial containing 75 μL of 5 μM each primer specific for the human GAPDH promoter. 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.

Analysis Note

Control
Includes negative control supernatant 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.