ChIPAb+ Monomethyl-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+ Monomethyl-Histone H3 (Lys4) set includes the Monomethyl-Histone H3 (Lys4) antibody, normal mouse IgG, and qPCR primers which amplify a 213 bp region of human GAPDH coding region. The Monomethyl-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 Monomethyl-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. The N-terminal tail of histone H3 protrudes from the globular nucleosome core and can undergo several different types of epigenetic modifications that influence cellular processes. These modifications include the covalent attachment of methyl or acetyl groups to lysine and arginine amino acids and the phosphorylation of serine or threonine.

Specificity

This antibody detects Monomethyl-Histone H3 methylated at Lys4.

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

Immunogen

Epitope: a.a. 1-12

Peptide corresponding to Histone H3 containing the sequence [ART(me1-K)QTARKSTGC] on which Lys4 is acetylated.

Application

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µg of either Normal Mouse IgG , or 2 µg Anti-Monomethyl-Histone H3 (Lys4)and the Magna ChIP^® G Kit (Cat. # 17-611). Successful immunoprecipitation of Monomethyl-Histone H3 (Lys4) associated DNA fragments was verified by qPCR using ChIP Primers GAPDH Coding Region as a positive locus, and GAPDH promoter primers (22-004) 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 G (Cat. # 17-409) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.
Specificity data:
HeLa acid extracts were resolved by electrophoresis, transferred to PVDF, and probed with anti-Monomethyl-Histone H3 (Lys4) (1:5000 dilution).To demonstrate specificity, antibody was preincubated with modified histone peptides:
Lane 1 No peptide
Lane 2 unmodified Histone H3
Lane 3 Monomethyl Histone H3 (Lys4)
Lane 4 Dimethyl Histone H3 (Lys4)
Lane 5 Trimethyl Histone H3 (Lys4)
Lane 6 Trimethyl Histone H3 (Lys9)
Lane 7 Trimethyl Histone H3 (Lys27)
Lane 8 Trimethyl Histone H4 (Lys20)
Proteins were visualized using donkey anti-mouse IgG conjugated to HRP and a chemiluminescence detection system.

Research Category
Epigenetics & Nuclear Function

Research Sub Category
Chromatin Biology

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

Packaging

25 assays per set. Recommended use: 2 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 2 µg of either Normal Mouse IgG, or 2 µg Anti-monomethyl-Histone H3 (Lys4) and the Magna ChIP^® G Kit (Cat. # 17-611). Successful immunoprecipitation of Monomethyl-Histone H3 (Lys4) associated DNA fragments was verified by qPCR using ChIP Primers GAPDH Coding Region (Figure 1).
Please refer to the EZ-Magna ChIP G (Cat. # 17-409) or EZ-ChIP (Cat. # 17-371) protocol for experimental details. (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

Anti-Monomethyl-HisPtone H3 (Lys4) (mouse monoclonal), One vial containing 50 µg of protein G purified antibody in 50 µL PBS containing 0.05% sodium azide. Store at -20°C.
Normal Mouse IgG, . Two vials containing 25 µg purified Mouse IgG in 25 µL storage buffer containing 0.1% sodium azide. Store at -20°C.
ChIP Primers, human GAPDH coding region, . One vial containing 75 μL of 5 μM each primer specific for human GAPDH coding region.
Store at -20°C.
FOR: GGC TCC CAC CTT TCT CAT CC
REV: GGC CAT CCA CAG TCT TCT GG

Format: Purified

Protein A purified

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 normal mouse IgG and primers specific for human GAPDH coding region.

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.