Anti-dimethyl Arginine 抗体 [21C7] (ab413)

Reviews (1)Q&A (9)References (7)

製品の概要

  • 製品名
    Anti-dimethyl Arginine antibody [21C7]
  • 製品の詳細
    Mouse monoclonal [21C7] to dimethyl Arginine
  • 由来種
    Mouse
  • 特異性
    This antibody reacts with free and bound asymmetric NG-NG-dimethyl arginine. It is not known whether the antibody reacts with symmetric dimethyl arginine.
  • アプリケーション
    適用あり: IP, WB, ChIPmore details
  • 免疫原

    Asymmetric NG -N G -dimethyl arginine (two methyl groups on one of the terminal nitrogen atoms of the guanidinium group)

  • 特記事項

    This antibody will be of use in chromatin and transcription factor research to monitor methylation status. Since it is specific to dimethyl arginine it can be used in parallel with ab412 (detects monomethyl and dimethyl arginine) and ab414 (detects monomethyl arginine only) to monitor the exact modification status.

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab413 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 dilution.
WB 1/50 - 1/500.
ChIP Use at an assay dependent concentration. Use 5µl for 4x106 cells.

ターゲット情報

  • 別名
    • Di methyl Arginine antibody

参考文献

This product has been referenced in:
See all 7 Publications for this product

ab413 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 10 Abreviews or Q&A

Question

Our customer wants to find antibodies specific for asymmetrical methyl arginine and specific for symmetirical antibody respectly. So please let me know this antibody can detect only asymmetrical methyl arginine or not. If you know specific antibody please let me us.

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Answer

Thank you for your enquiry.

I can confirm that ab412 mono and dimethyl Arginine antibody [7E6] cross reacts with both symetric and assymetric methyl arginine. However, the ab413dimethyl Arginineantibodyis specific to ADMA only.

https://www.abcam.com/index.html?datasheet=413 (or use the following: https://www.abcam.com/index.html?datasheet=413).

I hope this will be helpful to you. If you have any further questions, please do not hesitate to let me know.

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Question

samples; HEK293 cells block: 5% milk ab413, ab415: black membrane, protein ladder in white Ab 1/1000 suggestion: use less primary and/or secondary antibody ab109451: lots of bands, Ab 1/1000 suggestion: use less primary antibody (recommended dilution is 1/10,000 to 1/50,000)

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Answer

Thank you for your phone call. I am glad to hear that for ab109451 the protocol tips did work. I am very sorry that unfortunately, for ab413 and ab415, the tips did not improve your results. However, the details provided enable us to closely monitor the quality of our products. I am sorry these 2 products did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued free of charge replacements with the order number xxx for ab107495 and ab107514. You should receive both vials tomorrow. 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

samples; HEK293 cells block: 5% milk ab413, ab415: black membrane, protein ladder in white Ab 1/1000 suggestion: use less primary and/or secondary antibody ab109451: lots of bands, Ab 1/1000 suggestion: use less primary antibody (recommended dilution is 1/10,000 to 1/50,000)

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Answer

Thank you for your phone call. I am sorry to hear that these 3 antibodies are not providing satisfactory results. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. As we discussed over the phone, I would like to offer some suggestions to help optimize the results: For ab413, ab415 I'd suggest using less primary and/or secondary antibody as the membrane shouldn't turn black. This might be an indication of too much antibody on the membrane. For ab109451, most likely using less primary antibody could help with the background as the recommended dilution for the antibody is 1/10,000 to 1/50,000. Should the suggestions not improve the results, please do let me know. In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund. I hope this information is helpful, and I thank you for your cooperation.

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ChIP abreview for Anti-dimethyl Arginine antibody [21C7] - ChIP Grade

 Excellent
Abreviews
Application
ChIP
Sample
Human Cell lysate - nuclear (HL-60 cells)
Specification
HL-60 cells
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 0 second(s)
Specification of the cross-linking agent: formaldehyde
Detection step
Taqman
Positive control
input DNA
Negative control
No antibody controls.
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Dr. Balint Laszlo Balint

Verified customer

投稿 Oct 04 2005

Question

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 100537 DESCRIPTION OF THE PROBLEM I ordered three antibodies, ab412, ab413, and ab414. I am currently trying to optimize the conditions for Western blotting using these antibodies. None of these antibodies are able to immunostain my protein of interest, which is ICP27 otherwise known as IE63 from the herpes simplex type 1 virus. Published literature shows that this protein is known to be methylated. SAMPLE A total of 2*10^7 HeLa cells were infected with wild-type HSV-1 virus. Cells were harvested 5 hours post-infection. Nuclear and cytoplasmic fractions were separated. Our protein of interest, ICP27, was immunoprecipitated with our monoconal antibody H1119. The immunoprecipitate was resolved using 12% SDS-PAGE, transferred o/n to nitrocellulose, and Western analysis was done on 4 parallel blots using our monoclonal antibody H1119, and ab 412, ab413, and ab414. I have tried running crude extracts on a 12% SDS-PAGE gel, transferring to nitrocellulose, and Western blotting using H1119, which is a monoclonal antibody toward ICP27, as well as ab412, ab413, and ab414. These were done on parallel blots. PRIMARY ANTIBODY mab 412 (abcam) mouse monoclonal IgG mab 413 (abcam) mouse monoclonal IgM mab 414 (abcam) mouse monoclonal IgG diluted at (see concentrations we have tried below) into 0.3%BSA/0.1% Tween/PBS incubate at room temp on rotator for 1 hour wash 15 min, 5 min, 5 min with 0.1%Tween/PBS add secondary antibody DETECTION METHOD SuperSignal West Pico chemiluminescent substrate from Pierce POSITIVE AND NEGATIVE CONTROLS USED + controls: we run crude extracts in parallel and detect a bunch of proteins. we also are sure that ICP27 is present in the extracts, because we run a parallel Western using a stellar monoclonal antibody against ICP27. We see ICP27 in all samples we test; however, your antibodies do not show staining to ICP27 even though we know it is methylated in vivo. ANTIBODY STORAGE CONDITIONS Anitbody was reconstituted in 200 uL ddH20. Separated into 40 uL aliquots and stored at -20C. SAMPLE PREPARATION Protease inhibitors used = leupeptin and pefabloc buffers = immunoprecipitates were washed in hi salt lysis buffer followed by PBS, SDS-PAGE loading buffer was added to immunopreciptates (+10% beta mercaptoethanol, and boiled 5 min. AMOUNT OF PROTEIN LOADED Crude extract: 5% of the total nuclear or cytoplasmic HeLa extract taken from 2*10^7 cells. IP: approx. 5-10 ug ICP27 per lane ELECTROPHORESIS/GEL CONDITIONS 12% SDS-PAGE TRANSFER AND BLOCKING CONDITIONS Transfer buffer: 300 mL 10X Tris-glycine 600 mL methanol 2100 mL ddH2O transfer o/n our MW markers are colored, so we know the transfer are successful when the markers transferred, this step is never a problem for us block in 5% BSA/0.1%Tween PBS 1 hr. wash 15 min, 5 min, 5 min in 0.1%Tween PBS 1 hr. add primary antibody SECONDARY ANTIBODY Manufacturer: [a competitor] Species: anti-mouse IgG (for H1119, ab412, ab414) anti-mouse IgM (for ab413) diluted at 1:100,000 into 0.3%BSA/0.1% Tween/PBS incubate at room temp on rotator for 1 hour wash 15 min, 5 min, 5 min with 0.1%Tween/PBS add ECL enhancer and substrate HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 WHAT STEPS HAVE YOU ALTERED? amount of primary antibody (ab 412, 413, 414) used in WB. Crude hela extracts (nuc & cyto fractionated): WB Attempt #1 ab412 1:5000 did not work ab413 1:500 did not work ab414 1:500 did not work WB Attempt #2 ab412 1:250 worked ab413 1:250 worked ab414 1:250 worked Immunoprecipitated ICP27 from HSV1-infected HeLa extracts WB Attempt#1 ab412 1:250 did not work, did not see ANY bands except heavy & light chains ab413 1:250 did not work, did not see ANY bands except heavy & light chains ab414 1:250 did not work, did not see ANY bands except heavy & light chains WB attempt #2 ab412 1:100 no bands, except for heavy and light chains ab413 1:100 no bands, except for heavy and light chains ab414 1:100 no bands, except for heavy and light chains ADDITIONAL NOTES I am still trying to optimize my conditions and already I am running out of antibody. Will I continue to have to repeat these optimization conditions with every batch of ab412, 413, 414 I receive from your company? We know this protein is methylated, it is published data, which I have repeated several times in my lab. Background staining is not the problem, the problem is that your abs are not staining my protein of interest even though we know it is methylated.

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Answer

Thank you for your enquiry. Thank you also for completing our questionnaire so comprehensively. I am sorry that you have been having difficulties with our antisera against methyl arginine. I have examined your questionnaire and it seems as if you are following a protocol not dissimilar to Abcam's recommended protocol. You mentioned that your target protein is methylated. I have discovered that ICP27 is methylated at Arginine residues. I would have mentioned that your immunoprecipitation by H1119 may be obscured by the methylated form of the protein, resulting in the preferential precipitation of the unmethylated form. However, this does not explain the fact that you are NOT obtaining a result following western blotting using a crude HeLa extract. Given a crude extract the very least that I would expect to see would be the arginine methylated histones H3 and H4. In fact this is what I would recommend you use as positive control to guarantee that your abcam antisera are working and detecting methyl-arginine. Preparing a high concentrated histone extract can be done on HeLa cells. We have a very easy protocol at the link below. This will not affect your protocol given that you are using a 12.5% gel. Histones are between 10 and 17KDa. https://www.abcam.com/index.html?pageconfig=view_protocol&pid=316 You mention that you did have some success using a 1:250 dilution. Please can you e-mail me these scanned as JPeGs. I would also like to know the mass of protein you are loading onto the gel during the whole cell extract western blot and the relative amount you are loading following IP. I would recommend a starting amount of 20ug for the crude extract. Following some literature searching I would also like to know whether your ICP27 construct that you are injecting contains an RGG box as I have learnt that the methylation of ICP27 is dependent on its presence. I look forward to your reply and hope that we can resolve this issue quickly.

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Question

Concerning ab415, ab414, ab413 - says whole antiserum; is it tisue culture supernatant or ascites? Do ab412 and ab413 cross-reat with symmetrical Ng/Na - dimethyl arginine?

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Answer

Thank you for your patience. Ab415, ab414, and ab413 are ascites. It has not been determinded as to whether ab412 and ab413 cross-react with symmetrical Ng/Na - dimethyl arginine.

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Question

I recently purchased an Ab 413, the anti-dimethyl Arginine antibody.  There is no information regarding concentration of this antibody in the product information sheet, and while I realize a specfic concentration may not be known or tested, I am wondering if you can give me some information regarding concentration/ a concentration range of the Ab.

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Answer

Thank you for your enquiry and your interest in our products. We are sorry to inform you that there is no protein concentration for polyclonal antibodies that are not affinity purified. This antibody is provided as whole serum and the IgG concentration has not been determined.

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Question

I am interested in setting up an ELISA to quantitate serum concentrations of asymmetric dimethylarginine. However, I am concerned about cross-reactivity with free symmetric dimethylarginine. Do you have updated information on the extent of cross reactivity of ab413 with symmetric dimethylarginine? Has ab413 been successfully used in ELISA previously? Thanks for your assistance.

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Answer

This antibody has not been tested in ELISA. All the latest information that we have about this antibody is found on the datasheet.

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Question

Has this Ab been shown to react with diMeArg in a peptide when the surrounding sequence is very different from the sequence used to generate the Ab? If so, how many different sequences have been tested? Without information on the context dependency, I can't use the product.

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Answer

The epitope is di-methyl arginine independent of the surrounding sequence and has been used on numerous different diMeArg peptides.

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Question

What peptide sequence was used as the antigen?

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Answer

Sorry, but the immunogen of this antibody is confidential. All we can say is that it contained methylated arginine (the antibody is inhibited by the modified amino acid).

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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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