ChIPAb+ Dimethyl-Histone H3 (Lys4)

ChIPAb+ Dimethyl-Histone H3 (Lys4)

Stock Code: 3586470
Manufacturer Part No: 17-677
Order Now for 7 day delivery
Click Add to Quote for price and delivery
Quantity: - +

Analysis Note


ControlIncludes negative control mouse IgG antibody and control primers specific for human β-globin promoter.


Application


Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (2 X 106 cell equivalents per IP) was subjected to chromatin immunoprecipitation using 2 µg of either normal mouse IgG, or Anti-dimethyl-Histone H3 (Lys4) antibody and the Magna ChIP G Kit (Cat. #17-611). Successful immunoprecipitation of dimethyl-histone H3 (Lys4)-associated DNA fragments was verified by qPCR using β-globin ChIP Primers versus GAPDH Coding primers (Please see figures). Data is presented as percent input of each IP sample relative to input chromatin for each antibody with the indicated primers.
Please refer to the EZ-Magna G ChIP™ (Cat. #17-408) or EZ-ChIP™ (Cat. #17-371) protocol for experimental details.

ChIP-seq Analysis:
Chromatin immunoprecipitation was performed using the Magna ChIP™ HiSens kit (cat# 17-10460), 2 µg Anti-dimethyl-Histone H3 (Lys4) antibody (cat# 17-677), 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 sixteen 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. The highest 25% of peaks identified in the 04-790 and 17-677 datasets showed 92 and 90% overlap with peaks identified in the ENCODE H3K4me2 BROAD Histone track for HeLa S3.

Western Blot Analysis:
Recombinant Histone H3 (Lane 1) and HeLa acid extract (Lane 2) were resolved by electrophoresis, transferred to PVDF membrane and probed with Anti-dimethyl Histone H3 (Lys4) (2 μg/mL).
Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP (Cat. #AP124P) and a chemiluminescence detection system.

Dot Blot Analysis:
Absurance Histone H3 Antibody Specificity Array (Cat. No. 16-667) and Absurance Histone H2A, H2B, H4 Antibody Specificity Array (Cat. No. 16-665), which contain histone peptides with various modifications were probed with Anti-dimethyl H3 (Lys4) at 2.0ug/mL (1:500) dilution. Proteins were visualized using a Donkey anti-mouse IgG conjugated to HRP and a chemiluminescence detection system.

Beadlyte™ Histone Peptide Specificity Assay:
0.15 μg/ml of purified anti-dimethyl-Histone H3 (Lys4),
clone CMA303 was incubated with a cocktail of microspheres
conjugated to histone H3 peptides with the following modifications:
1. unmodified H3 (K4)
2. monomethyl H3 (K4)
3. dimethyl H3 (K4)
4. trimethyl H3 (K4)
5. trimethy H3 (K27)
Unbound antibody was then removed by filtration.
Peptide antibody complexes were incubated with a
PE-conjugated anti-mouse secondary antibody.

Quality Level100
ManufacturerSIGMA-ALDRICH

There are no downloads for this product.