Anti-mono and dimethyl Arginine 抗体 [7E6] (ab412)

Reviews (1)Q&A (39)References (31)

製品の概要

  • 製品名
    Anti-mono and dimethyl Arginine antibody [7E6]
  • 製品の詳細
    Mouse monoclonal [7E6] to mono and dimethyl Arginine
  • 由来種
    Mouse
  • アプリケーション
    適用あり: IP, ChIP, ELISAmore details
    適用なし: WB
  • 種交差性
    交差種: Species independent
  • 免疫原

    Asymmetrical NG/NG-dimethyl arginine

  • ポジティブ･コントロール
    • Methylated histones are an option
  • 特記事項

    This antibody will be of central importance in analysing the methylation status of chromatin and transcription factors. Since it recognises both dimethyl and monomethyl arginine it can be used in parallel with ab413 (detects dimethyl arginine only) and ab414 (detects monomethyl arginine only) to monitor the exact modification status. A tissue culture supernatant version is available as ab5394.

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab412 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 3µl for 106 cells.
ELISA Use a concentration of 0.05 - 0.5 µg/ml.
  • 追加情報
    Is unsuitable for WB.

    • ターゲット情報

    • 別名
      • Di methyl Arginine antibody
      • Dimethyl Arginine antibody
      • Methyl Arginine antibody
      • Mono methyl Arginine antibody
      • Monomethyl Arginine antibody
      see all

    画像

    • ChIP - Anti-mono and dimethyl Arginine antibody [7E6] (ab412)
      ChIP - Anti-mono and dimethyl Arginine antibody [7E6] (ab412)This image is courtesy of Sylvain Daujat, Tony Kouzarides lab, Cambridge University, UK

      Sonicated Chromatin prepared from untreated or 17beta-estradiol (E2) treated MCF7 cells was subjected to the ChIP procedure (see protocol) with ab412 to mono and dimethyl Arginine.

      Immunoprecipitated chromatin was analysed in the proximal region of the estrogen-responsive pS2 promoter (as shown above) and quantified by real-time PCR (values are % of inputs). The primers are designed to follow the nucleosome E (including the Estrogen Responsive Element ERE). 3 µl of ab412 and 4x106 cells were used in each ChIP experiment.

    • Immunoprecipitation - Anti-mono and dimethyl Arginine antibody [7E6] (ab412)
      Immunoprecipitation - Anti-mono and dimethyl Arginine antibody [7E6] (ab412)Image from P Gupta et al, PLoS ONE 3:e2658 (2008), Fig 2.
      RIP140 null adipocytes extracts were suspended in 250 µl of immunoprecipitation buffer (150 mM NaCl,50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.2% (v/v) Nonidet P40, 2 mM PMSF, 0.1% (w/v) SDS and a protease-inhibitor cocktail. Protein extracts (200 µg) were incubated with antibodies to RIP140, 14-3-3, or PRMT1 overnight at 4°C, and precipitated with protein G–agarose beads for 1–2 hours. ab412 was then used (amongst other antibodies) in Western Blot. Reduced protein inputs were used for ectopic expression of RIP140 to avoid saturation.

    参考文献

    This product has been referenced in:
    • Ganesan M  et al. Demethylase JMJD6 as a New Regulator of Interferon Signaling: Effects of HCV and Ethanol Metabolism. Cell Mol Gastroenterol Hepatol 5:101-112 (2018). Read more (PubMed: 29693039) »
    • Kong GM  et al. Selective small-chemical inhibitors of protein arginine methyltransferase 5 with anti-lung cancer activity. PLoS One 12:e0181601 (2017). Read more (PubMed: 28806746) »
    See all 31 Publications for this product

    ab412 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。

    レビューと Q&A

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    1-10 of 40 Abreviews or Q&A

    Immunoprecipitation abreview for Anti-mono and dimethyl Arginine [7E6] antibody

     Excellent
    Abreviews
    abreview image
    Application
    Immunoprecipitation
    Immuno-precipitation step
    Protein A/G
    Sample
    Human Cell lysate - whole cell (fibroblast)
    Specification
    fibroblast
    Total protein in input
    1000 µg
    Read More

    Abcam user community

    Verified customer

    投稿 Jun 19 2014

    Question

    I need Discount code for the three below antibodies . Please arrange to send the same at your earliest .

    ab21623, ab23366 and ab412

    for detection of methylation and acetylation of bacterial proteins.

    Regards

    Read More
    Answer

    Thank you for contacting us.

    These antibodies are raised against targets that are not species specific. The antibodies would detect the proteins irrespective of species so the Abtrial discount will not be valid.

    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Read More

    Question

    Dear Kate,

    thank you for your email. I would appreciate if you could send me a free of charge replacement.

    Thank you very much.

    Read More
    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 #######.

    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.

    Read More

    Question

    1. Could you confirm if reducing and denaturing conditions have been tried? We recommend to use antibodies in reducing and denaturing conditions in WB unless it recommends to use non denaturing and reducing conditions on the datasheet. This will ensure that the proteins are in the correct conformation to run at the correct molecular weight and be detected by the antibody. Samples were run in reducing and denaturing conditions using SDS and DTT.

    2. Could you confirm what size are the 3 bands in the blot in the eluted samples? And what size bands were you expecting? The bands in lane 3 are presumably the eluted antibody:˜120 kDa, ˜50 kDa and ˜24kDa

    3. How was the sample eluted from the IP column? Which buffer was used? Samples were eluted by boiling in denaturing SDS sample buffer.
    4. Which lanes are the whole cell extract in, is this lane 1 and 2 (input A and B?). Yes. These lanes should give various signals, since I doubt that there is no methylated protein in the extract.

    5, Has the transfer to the membrane and quality of the sample been assessed with a loading control in the whole cell extracts? Transfer to the membrane may be different from the eluted samples if they are in different buffers etc). Yes, there were more samples on the gel and these blotted and performed just fine with the respective antibodies in all cases. Blotting was also confirmed by Ponceau staining.

    Read More
    Answer

    Thank you for your message and for providing this further information.

    I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments and would be pleased to arrange a free of charge replacement or credit note in compensation.

    I look forward to hearing from you with details of how you would like to proceed.

    Read More

    Question

    Dear Madam/Sir,

    please see below. I don’t think the antibody ab412 works correctly.

    Kind regards,



    Order Details
    Antibody code: ab412

    Problem
    Choose: No signal

    Lot number GR104284-1

    Purchase order number
    or preferably Abcam order number:
    ######

    General Information
    Antibody storage conditions (temperature/reconstitution etc) -20°C


    Description of the problem no signal in whole cell extracts.


    Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)
    N2a cells (murine cell line), whole cell extract

    Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)
    Lysis in 50 mM Tris-HCl 8,0 + 140 mM NaCl + 0,25% NP40 + protease inhibitor mix
    Sample preparation: 10 µl sample + 4,7 µl Reducing loading buffer (Fermentas), 5 min 95°C


    Amount of protein loaded
    ˜100 µg

    Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
    10% denaturing, non-reducing gel

    Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)
    Tank Blotting 1 h 150 mA (Biorad)

    Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
    1:1000 ab412 in PBS/milk

    Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
    1:10.000 anti-mouse-POD (A4416, Sigma)


    Detection method (ECL, ECLPlus etc.)
    ECL-Plus (Perkin Elmer)

    Positive and negative controls used (please specify)
    n/a


    Optimization attempts (problem solving)
    How many times have you tried the Western?
    3 times


    Have you run a "No Primary" control?
    Yes No Yes

    Do you obtain the same results every time?
    Yes No Yes
    e.g. are the background bands always in the same place?


    What steps have you altered?
    different amount of protein used for IP / input

    More antibody ab412 used for IP

    Additional Notes:


    Image:
    We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to assess the results.
    see attached file, lane 1, input A; Lane 2, input B; lane 3, eluate A; lane 4, eluate B

    Read More
    Answer

    Thank you for taking the time to complete our questionnaire. The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

    Reviewing this case, I would appreciate if you can confirm some further details:

    1. Could you confirm if reducing and denaturing conditions have been tried? We recommend to use antibodies in reducing and denaturing conditions in WB unless it recommends to use non denaturing and reducing conditions on the datasheet. This will ensure that the proteins are in the correct conformation to run at the correct molecular weight and be detected by the antibody.

    2. Could you confirm what size are the 3 bands in the blot in the eluted samples? And what size bands were you expecting?

    3. How was the sample eluted from the IP column? Which buffer was used?

    4. Which lanes are the whole cell extract in, is this lane 1 and 2 (input A and B?).

    5, Has the transfer to the membrane and quality of the sample been assessed with a loading control in the whole cell extracts? Transfer to the membrane may be different from the eluted samples if they are in different buffers etc).

    I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

    Read More

    Question

    ab412 no signal in WB
    Using M2A mouse neuroblastoma cell line

    Read More
    Answer

    Thank you for your recent telephone call and for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

    I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for WB. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

    As requested, I have obtained some more information regaring Western blot protocols using ab412 mono and dimethyl Arginine antibody [7E6] . I hope the following publications in which the antibody has been used in WB will be helpful:

    Blifernez O et al. Protein arginine methylation modulates light-harvesting antenna translation in Chlamydomonas reinhardtii. Plant J 65:119-30 (2011).

    McBride AE et al. Specific sequences within arginine-glycine-rich domains affect mRNA-binding protein function. Nucleic Acids Res 37:4322-30 (2009).

    Gupta P et al. PKCepsilon stimulated arginine methylation of RIP140 for its nuclear-cytoplasmic export in adipocyte differentiation. PLoS ONE 3:e2658 (2008).

    As discussed on the telephone, I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

    I would appreciate if you could also provide an image which would help us to assess the results.

    Thank you for your time and cooperation. We look forward to receiving the completed questionaire.



    Order Details
    Antibody code:

    Problem
    Choose: Non-specific band Multiple bands No signal or weak signal High background

    Lot number

    Purchase order number
    or preferably Abcam order number:



    General Information
    Antibody storage conditions (temperature/reconstitution etc)


    Description of the problem (high background, wrong band size, more bands, no band etc.)


    Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)


    Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)


    Amount of protein loaded


    Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)


    Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


    Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


    Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


    Detection method (ECL, ECLPlus etc.)


    Positive and negative controls used (please specify)



    Optimization attempts (problem solving)
    How many times have you tried the Western?



    Have you run a "No Primary" control?
    Yes No

    Do you obtain the same results every time?
    Yes No
    e.g. are the background bands always in the same place?


    What steps have you altered?


    Additional Notes:


    Image:
    We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

    Read More

    Question

    I noticed that Abcam has in its inventory anti mono- and di-methyl arginine antibody (ab412), and anti-methylated lysine (mono-methyl, dimethyl) antibody (ab23366).
    Could you please send us a small sample of the antibodies mentioned above for a test in our in vitro system? Of course whichever of those antibodies that gives us a positive result will be ordered from you in the future.
    Thank you so much for your time and consideration.

    Read More
    Answer

    Thank you for contacting us. Because we carry over 90,000 products, it isn't feasible for us to keep small sample sizes of our products.

    We are happy to reassure our customers that all of our products are covered by our Abpromise, which guarantees that the product will work in the applications and species specified on the datasheet, or we will offer a replacement, credit, or refund within 6 months of purchase.

    Otherwise, we like to encourage all of our customers to submit an Abreview via the online product datasheet. We always appreciate customer feedback, whether positive or negative, and we make all product information available to researchers. Plus, each Abreview earns Abpoints that can be used for discounts on future purchases or rewards such as Amazon.com gift certificates.

    To find out more about our Abreview system, please see the following link:

    https://www.abcam.com/abreviews

    I hope this information is helpful. Please do not hesitate to contact us again with any other questions.

    Read More

    Question

    Thank you.
    We prefer to get refund.
    Please advise how to proceed to receive the refund.

    Read More
    Answer

    Thank you for contacting us.
    Your credit note ID is *.
    I am sorry that this antibody did not perform as stated on the datasheet. If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used.
    Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department.
    The credit note ID is for your reference only and does not automatically guarantee the credit.
    I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

    Read More

    Question

    Please see our reponse. Thanks.
    1 - What sample type are you using? : we tried 293T cell lysates and mouse tissue (liver) lysates prepared in RIPA buffer.
    2 - Are you seeing any bands, no bands etc?: no clear bands, there are a few very faint ones after long exposure
    3 - How much sample are you loading? : we tried from 20 to 100ug per lane
    4 - What type of blocking agent are you using? : we use 5% non fat dry milk in TBST.
    5 - What primary antibody concentrations have you tried and how long do you incubate for?: we tried 1:500 ˜ 1:1000 in 1% BSA in TBS, incubated overnight (16hr) at 4 degree with rocking motion.

    Read More
    Answer

    Thank you for your reply.
    I am sorry that the antibody is not working for you an based on the information that you have provided, I do not believe that there is ay protocol advice that I can offer that would resolve the issue.
    Therefore I would like to offer either a replacement antibody or a refund. A possible aletrantive could be:
    https://www.abcam.com/mono-and-dimethyl-Arginine-antibody-7E6-ab5394.html
    Please let me know how you would like to continue.
    I look forward to your reply.

    Read More

    Question

    We have purchased below antibody (ab412 Mouse monoclonal [7E6] to mono and dimethyl Arginine)and tried it for WB application as recommended by the company. So far there is absolutely no signal we could detect under different conditions using various samples. We strongly suspect that it is a matter of primary antibody. We normally use methionine depleted media-fed whole cell lysate as controls.
    Please let me know how to trouble shoot or even receive a replacement for the product. Shipment condition was fine and another antibody from Abcam that arrived on the same day perform fine.

    Read More
    Answer

    Thank you for contacting Abcam.
    The antibody is covered under our Abpromise for six months and is guaranteed to work in western blot on a variety of samples . If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.
    To be able to help further in this matter, could you please provide me some more information about the nature of the problem and also some protocol information?
    1 - What sample type are you using?
    2 - Are you seeing any bands, no bands etc?
    3 - How much sample are you loading?
    4 - What type of blocking agent are you using?
    5 - What primary antibody concentrations have you tried and how long do you incubate for?
    We do take all complaints on products seriously and we do our best to solve these problems.

    Read More

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

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