Anti-pan methyl Lysine 抗体 - ChIP Grade (ab7315)

Reviews (2)Q&A (12)References (21)

Key features and details

  • Rabbit polyclonal to pan methyl Lysine - ChIP Grade
  • Suitable for: IP, ChIP, ELISA, WB, ICC/IF, IHC-P
  • Reacts with: Species independent
  • Isotype: IgG

製品の概要

  • 製品名

    Anti-pan methyl Lysine antibody - ChIP Grade

  • 製品の詳細

    Rabbit polyclonal to pan methyl Lysine - ChIP Grade

  • 由来種

    Rabbit

  • 特異性

    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.

  • アプリケーション

    適用あり: IP, ChIP, ELISA, WB, ICC/IF, IHC-Pmore details

  • 種交差性

    交差種: Species independent

  • 免疫原

    Full length native protein (purified) corresponding to Cow pan methyl Lysine.

  • 特記事項

    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.

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

製品の特性

アプリケーション

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.

アプリケーション Abreviews 特記事項
IP Use at an assay dependent concentration.
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.
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.

ターゲット情報

  • 関連性

    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.

画像

  • Western blot - Anti-pan methyl Lysine antibody - ChIP Grade (ab7315)
    Western blot - Anti-pan methyl Lysine antibody - ChIP Grade (ab7315)
    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).
  • ChIP - Anti-pan methyl Lysine antibody - ChIP Grade (ab7315)
    ChIP - Anti-pan methyl Lysine antibody - ChIP Grade (ab7315)

     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.

  • Immunoprecipitation - Anti-pan methyl Lysine antibody - ChIP Grade (ab7315)
    Immunoprecipitation - Anti-pan methyl Lysine antibody - ChIP Grade (ab7315)
    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.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pan methyl Lysine antibody - ChIP Grade (ab7315)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pan methyl Lysine antibody - ChIP Grade (ab7315)
    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.
  • Immunocytochemistry/ Immunofluorescence - Anti-pan methyl Lysine antibody - ChIP Grade (ab7315)
    Immunocytochemistry/ Immunofluorescence - Anti-pan methyl Lysine antibody - ChIP Grade (ab7315)

    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.

プロトコール

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.

Click here to view the general protocols

参考文献 (21)

ab7315 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

ab7315 は 21 報の論文で使用されています。

  • 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
View all Publications for this product

レビューと Q&A

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1-10 of 14 Abreviews or Q&A

Question

Customer called as they were seeing no bands using this product.

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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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Question

To whom it may concern,

Thank you very much for all your help in this matter. It is great working with a company that cares about customer concerns.
If possible could be be refunded for either one of the two we purchased, and for the other exchange it for ab23366, a mono/dimethyl lysine antibody.

I am not sure of the order # or date as our purchasing office has closed for the day. If you need more information please let me know and I will talk with them tomorrow. Otherwise, I hope this information is sufficient.

Thank you again for all your time and help.

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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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Question

To whom it may concern,

Thank you again for your reply. I will try to provide you with the answers to the questions.

Question 1. see figure below. Our lab has done several MYC IPs and have IB with MYC antibodies to confirm the presence of MYC. In the figure I attached (most recent IP) I IP MYC using MYC C-33 and IB with a acetylated MYC antibody. I further stripped and re-probed the blot with MYC N262 to further confirm the presence of MYC in the IP

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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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Question

To whom it may concern,

Thank you again for the fast reply. Here is the information you are asking for:

1. Yes, two different MYC antibodies for IP and a MAX antibody for a MYC co-iP
2. Myc band is typically seen about 62 to 65 kDa. For IP I run the gel much longer to avoid the MYC band from NOT separating from IgG band.
3. yes, we would like to only see bands in IP lane and not the control lane.


Thank you very much again for all your help and time.

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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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Question

Thank you very much for your reply and your concern and any help you may be able to provide in exchanging or providing the early stated new methyl antibody.
Here is the information you asked for:
Samples: HeLa lysates made from 400mM NaCl lysis buffer.
Application of abcam antibodies: detection of a PTM (methylation) on the MYC protein.
Brief protocol:
block nitrocellulose membrane after transfer with 6% NFD milk for 30m.
1. incubate in primary antibody (either ab412, 1:1K or ab7315 1:1K in +4/ON)
2. Wash away excess primary with TBST X 3, 5m each wash
3. incubate in secondary HRP linked antibodies (1h for normal secondary 1:5K, 30min for true blot 1:5K + dark)
4. wash away excess secondary antibody with TBST X3, 5m each wash.
5. use ECL X1 min, and expose to x-ray film.

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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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Question


I ordered two antibodies from your company (ab412, 200uL, $355. and ab7315, 50ug, $355). Both antibodies provided disappointing results. I am aware of a 3rd antibody ,ab23366, which I found referenced in "Genes and Development." Due to the large amount of money spent on the first two antibodies, and the lack of data obtained, I was wondering if we could try/exchange for this antibody or obtain a small sample for a single WB (using a 1:500 dilution, to make ˜40mL total antibody+milk mix). Thank you very much for any help you can provide.

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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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Question

I checked the antibody ab80182 Anti-Methylated Lysine antibody. I am confused about your webpage introduction of this antibody. In the datasheet, it is written that "ab76118 recognizes proteins with trimethylation on lysine residues (N-epsilon). It does not cross-react with acetylated proteins and mono and dimethylated proteins.", while in the section of "Scientific support", it is written that " This antibody specifically recognizes proteins that contain trimethylated lysines. It will cross react with acetylated proteins, mono-methylated proteins, or di-methylated proteins". Which is right?? I have another question. If you don't mind, would you please explain why you do not mentioned the antibody:ab7315 pan methyl Lysine (methyl K pan) antibody? According to the introduction, it can be used to do IP. Sorry for so many questions. Thank you very much.  

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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

Hi, ab7315 and ab76118. Thanks,

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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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Western blot abreview for Anti-pan methyl Lysine antibody - ChIP Grade

 Average
Abreviews
abreview image
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
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Abcam user community

Verified customer

投稿 Nov 20 2009

Question

Cindy I've tried again with more protein, but unfortunately no success still. Would it be possible to exchange the antibody with another one? I notice you have another methyl lysine targetting antibody (ab23366). Could a swap for this one be arranged? >

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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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