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

Customer reviews and Q&As

1-10 of 14 Abreviews or Q&A

Question
Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products. I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number XXXXX. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.

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Answer

As both antibodies failed to work in western blot, I have sent you ab23366 as a free of charge replacement for ab7315, your new order number is ******** andI have also credited the cost of ab412, your credit note number is **********.

I am sorry about the problems you had with these antibodies and I hope that ab23366 works out for you. If there is anything else I can do, please let me know.

Hope you have a good evening.

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Answer

I really appreciate you sending that further data.

Since it is clear that ab412 and ab7315 are not detecting your protein of interest in your western blot and under our Abpromise, we guarantee that both should work, then I would be very happy to either send you replacement antibodies or to process a refund.

Please let me know how you would like to proceed and if you would like to receive replacements antibodies, let me know the catalogue numbers of those you would like. Could you also provided me with the original order details for these antibodies (either order #, purchasing agent name, date of purchase) so that I can complete your request.

Thank you once again for your patience and I look forward to your reply.

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Answer

Thank you for answering those questions and your continued patience.

Firstly, I would just like to reiterate that both ab412 and ab7315 are guaranteed to work in IP and so if we cannot determine what the problem is, then I will be happy to replace or refund these antibodies.

Having said that, I do have a few more questions/suggestions:

1 - Have you confirmed that the Myc antibodies are actually IP'ing your sample of interest. You could determine this, buy using the Myc N262 to IP and then blot with MycC33.

2 - Your input sample (you load 20ug?), does give a signal for your protein of interest, but have you tried starting with more total protein. I ask this as although you are using 20ug of total protein but increasing that amount (to say 40ug) you may be able to get more of your protein of interest to actually be IP'd and so get a signal using the antibodies.

I look forward to your reply and helping you resolve this issue.

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Answer

Thank you for providing that extra information.

Just to make sure that I understand your western blots:

1 - You are using a Myc antibody to do the IP, correct?

2 - What is the size of your band of interest? As you are seeing a band about ˜60kDa in your inout lanes and also seeing the same size band in your control and IP lane.

3 - Are you only expecting to see bands in your IP lane, NOT your control IgG lanes?

Thank you for your patience in this, I just want to make sure that I understand the problems that you are having, so that I can give you the best advice or send you the appropriate antibody.

I look forward to your reply.

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Answer

Thank you for contacting Abcam.

I am sorry that you had disappointing results using some of our products, under our Abpromise, we guarantee our products to work as stated on the datasheet. Since you have been having trouble with them, I will be happy to look over protocol that you have been using and see if there is any suggestions I can make. Could you please tell me the types of samples you are using, application antibody is being used in and also the general protocol you have used (including primary antibody concentration, incubation times, blocking buffers etc). Also, could you provide a description of the problem that you are having, or if you can send a copy of the results that you are seeing. We do take all complaints about our products seriously and do our best to resolve these issues.

I look forward to your reply and helping you resolve these issues.

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Answer

Thank you for your message. You mentions ab80182 as well as ab76118 at the beginning of this email. I assume you are meant to refer to datasheet of ab76118 only, not ab80182? I apologise for any confusion. I have been in contact wtih our source in order to clarify this information. They have confirmed we were regrettably originally sent incorrect information for the previous enquiry. I can confirm that this antibody will not cross react with acetylated proteins, mono-methylated proteins, or di-methylated proteins. We aim to provide accurate and correct information to customers, and I am sincerely sorry there was some confusion in this case. I have removed the published enquiry that had the incorrect details on the datasheet. With regards to ab7315, I am sorry this did not come up in my previous search on our system for you. I can confirm that this is tested in IP, and wil be suitable for this application. Thank you for your understanding. If you have any further questions, please do not hesitate to contact us. 

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Question
Answer

Thank you for your reply and for confirming the product numbers. I am pleased to provide the following information: ab76118 Methylated Lysine antibody: This antibody specifically recognizes proteins that contain trimethylated lysines. It will cross react with acetylated proteins, mono-methylated proteins, or di-methylated proteins ab7315 pan methyl Lysine (methyl K pan) antibody: This antibody has been tested with tri-methylated lysines (K4, K27 and K9) but it was found that the antibody recognizes these to a lesser extent than di-methylated lysine residues. This information is available on the datasheet underneath the western blot image. There is also a note in the specificity section stating that histone H3 tri-methyl K27 was recognized to a lesser extent in ELISA analysis. I hope this information will be helpful to you. Should you have any further questions, please do not hesitate to contact us.

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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

Answer

Thank you for contacting us. I am very sorry to hear you are still having a problem with ab7315 (pan methyl Lysine (methyl K pan) antibody - ChIP Grade). I'm also sorry I didn't ask earlier but could you clarify a couple of things for me: 1) You mentioned: "Bacterially expressed and purified GST-tagged protein (of human origin) which has been methylated in vitro using the methyltransferase SET 7/9" as well as "However, the antibody picked up protein whether it was methylated or not, suggesting that this low level binding was non-specific" but doesn't methylation take place in bacteria - are you using a mutant strain? Have you tried in vitro translation of histone - can you do that? 2) You mentioned: "Incubation overnight with the antibody improved the signal (but only on an overnight exposure to film). However, the antibody picked up protein whether it was methylated or not, suggesting that this low level binding was non-specific. did you detect improved signal with increased protein loading or no signal at all? I have confirmed your Abcam order 183065 (1X ab7315, lot 166243). Once I receive your reply and determine there is a problem with the antibody, I will be happy to replace ab7315 with ab23366 for you. I apologize for any inconveniences and I look forward to receiving your reply.

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1-10 of 14 Abreviews or Q&A

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