Product nameAnti-pan methyl Lysine antibody - ChIP Grade
DescriptionRabbit polyclonal to pan methyl Lysine - ChIP Grade
Specificityab7315 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 applicationsSuitable for: ChIP, ELISA, IP, WB, ICC/IF, IHC-Pmore details
Species reactivityReacts with: Species independent
Full length native protein (purified) corresponding to Cow pan methyl Lysine.
- 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.
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.
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Add glycerol to a final volume of 50% for extra stability and aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
Storage bufferpH: 7.40
Preservative: 0.02% Sodium azide
Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
Concentration information loading...
PurityImmunogen affinity purified
Primary antibody notesThis 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.
ChIP Related Products
- Human Histone H3 (mono methyl K4) peptide (ab1340)
- Human Histone H3 (mono methyl K9) peptide (ab1771)
- Human Histone H3 (di methyl K9) peptide (ab1772)
- Human Histone H3 (mono methyl K27) peptide (ab1780)
- Human Histone H3 (di methyl K27) peptide (ab1781)
- Human Histone H3 (tri methyl K27) peptide (ab1782)
- Human Histone H3 (unmodified ) peptide (ab2623)
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.
|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.|
RelevanceLysine 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.
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.
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.
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
ab7315 has been referenced in 21 publications.
- 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). 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). PubMed: 29974865
- A P et al. EZH2 promotes DNA replication by stabilizing interaction of POLd and PCNA via methylation-mediated PCNA trimerization. Epigenetics Chromatin 11:44 (2018). PubMed: 30071900
- Song H et al. Crosstalk between lysine methylation and phosphorylation of ATG16L1 dictates the apoptosis of hypoxia/reoxygenation-induced cardiomyocytes. Autophagy 14:825-844 (2018). PubMed: 29634390
- Sbirkov Y et al. Semi-Quantitative Mass Spectrometry in AML Cells Identifies New Non-Genomic Targets of the EZH2 Methyltransferase. Int J Mol Sci 18:N/A (2017). AP ; Human . PubMed: 28678185