Overview

  • Product name
    Anti-pan methyl Lysine antibody - ChIP Grade
  • Description
    Rabbit polyclonal to pan methyl Lysine - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    ab7315 recognises Histone H3 di-methyl K4, di-methyl K9 and di-methyl K27 in WB. Non-histone samples have not been tested, thus, we do not know if ab7315 would work on non-histone samples.
  • Tested applications
    Suitable for: ChIP, ELISA, IP, WB, ICC/IF, IHC-Pmore details
  • Species reactivity
    Reacts with: Species independent
  • Immunogen

    Purified calf thymus H1 chemically methylated on lysines. The reaction generates di-methyl lysine only.

    Read Abcam's proprietary immunogen policy

  • Positive control
    • This antibody gave a positive signal in calf thymus histone lysate and breast carcinoma formalin fixed parrafin embedded tissue This antibody gave a positive signal in the following Methanol fixed cell lines: HeLa.
  • General notes

    This antibody will be extremely useful in the study of the regulation of transcription by methylation. Has also been successfully used in CHIP in both human and yeast.

Properties

Applications

Our Abpromise guarantee covers the use of ab7315 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ChIP Use at an assay dependent concentration. Every new batch of this antibody is tested at Abcam in ChIP.
ELISA Use at an assay dependent concentration.
IP Use at an assay dependent concentration. PubMed: 20603076
WB 1/1000 - 1/2000. Predicted molecular weight: 14-17 kDa.
ICC/IF Use a concentration of 5 µg/ml.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

  • Relevance
    Lysine methylation occurs in three distinct states, having either one (me1), two (me2) or three (me3) methyl groups attached to the amine group of the lysine side chain. In eukaryotes, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with active chromatin and gene expression.

Images

  • All lanes : Anti-pan methyl Lysine antibody - ChIP Grade (ab7315) at 1 µg/ml

    Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate
    Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (unmodified ) peptide (ab7228) at 0.5 µg/ml
    Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.5 µg/ml
    Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K4) peptide (ab7768) at 0.5 µg/ml
    Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K4) peptide (ab1342) at 0.5 µg/ml
    Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.5 µg/ml
    Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.5 µg/ml
    Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.5 µg/ml
    Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K27) peptide (ab1780) at 0.5 µg/ml
    Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K27) peptide (ab1781) at 0.5 µg/ml
    Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K27) peptide (ab1782) at 0.5 µg/ml

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 14-17 kDa


    Exposure time: 20 minutes


    Expected molecular weights: H3 = 17 kDa; H4 = 14 kDa This image shows that the main epitopes recognized by ab7315 are the di methylated lysine residues. This can be seen in lanes 4, 7 and 10 where the activity of the antibody is quenched by the immunizing peptides (ab7768, ab1772, ab1781).
  •  Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 6.5µl of ab7315 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

  • pan methyl Lysine was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of it polyclonal to pan methyl Lysine and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab7315.
    Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
    Band: 17kDa: pan methyl Lysine.
  • IHC image of pan methyl Lysine (methyl K pan) staining in human breast carcinoma FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab7315, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
  • ICC/IF image of ab7315 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab7315 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:
  • Bueno MTD  et al. Recruitment of lysine demethylase 2A to DNA double strand breaks and its interaction with 53BP1 ensures genome stability. Oncotarget 9:15915-15930 (2018). Read more (PubMed: 29662616) »
  • Lee SW  et al. MicroRNAs Overcome Cell Fate Barrier by Reducing EZH2-Controlled REST Stability during Neuronal Conversion of Human Adult Fibroblasts. Dev Cell 46:73-84.e7 (2018). Read more (PubMed: 29974865) »
See all 19 Publications for this product

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1-2 of 2 Abreviews

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Recombinant protein (synthesis peptides)
Loading amount
0.1 µg
Specification
synthesis peptides
Gel Running Conditions
Non-reduced Non-Denaturing (Native) (dot blot)
Blocking step
Milk as blocking agent for 15 minute(s) · Concentration: 2% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Nov 20 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Arabidopsis thaliana Tissue lysate - whole (Whole plant tissue)
Specification
Whole plant tissue
Type
Native ChIP (N-ChIP)
Specification of the cross-linking agent: na
Detection step
Other
Positive control
Positive Knob180 (Z. Maize)
Negative centC (Z. Maize)

Abcam user community

Verified customer

Submitted Jan 12 2006

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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