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  1. Link

    rad17-phospho-s645-antibody-ab3620.pdf

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Epigenetics and Nuclear Signaling DNA / RNA DNA Damage & Repair DNA Damage Response DNA Damage Recognition
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Anti-Rad17 (phospho S645) 抗体 (ab3620)

  • Datasheet
  • SDS
Submit a review Q&A (7)References (4)

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Abpromise

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Western blot - Anti-Rad17 (phospho S645) antibody (ab3620)

    Key features and details

    • Rabbit polyclonal to Rad17 (phospho S645)
    • Suitable for: WB
    • Reacts with: Human
    • Isotype: IgG

    こちらの製品もご検討ください

    二次抗体
    Product image
    Goat Anti-Rabbit IgG H&L (HRP) (ab205718)

    関連製品

    製品の概要

    • 製品名

      Anti-Rad17 (phospho S645) antibody
      Rad17 一次抗体 製品一覧
    • 製品の詳細

      Rabbit polyclonal to Rad17 (phospho S645)
    • 由来種

      Rabbit
    • アプリケーション

      適用あり: WBmore details
    • 種交差性

      交差種: Human
      交差が予測される動物種: Chimpanzee, Rhesus monkey, Gorilla, African green monkey, Orangutan
    • 免疫原

      Synthetic phospho peptide (Human) - which represents a portion of human Rad 17around serine 645.

    • 特記事項

      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

    • 製品の状態

      Liquid
    • 保存方法

      Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
    • バッファー

      pH: 7
      Preservative: 0.1% Sodium azide
      Constituents: 0.021% PBS, 1.764% Sodium citrate, 1.815% Tris
    • Concentration information loading...
    • 精製度

      Immunogen affinity purified
    • 特記事項(精製)

      Antibodies that were not phospho-specific were removedby solid phase absorption. Antibodies specific for Rad 17pSer 645 were affinity purified using the phosphopeptideimmobilized on solid support.
    • ポリ/モノ

      ポリクローナル
    • アイソタイプ

      IgG
    • 研究分野

      • Epigenetics and Nuclear Signaling
      • DNA / RNA
      • DNA Damage & Repair
      • DNA Damage Response
      • DNA Damage Recognition

    関連製品

    • Compatible Secondaries

      • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
      • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Isotype control

      • Rabbit IgG, polyclonal - Isotype Control (ChIP Grade) (ab171870)
    • Recombinant Protein

      • Recombinant Human Rad17 protein (ab159302)

    アプリケーション

    Our Abpromise guarantee covers the use of ab3620 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
    WB 1/500 - 1/5000. Detects a band of approximately 85-88 kDa (predicted molecular weight: 77 kDa).

    ターゲット情報

    • 機能

      Essential for sustained cell growth, maintenance of chromosomal stability, and ATR-dependent checkpoint activation upon DNA damage. Has a weak ATPase activity required for binding to chromatin. Participates in the recruitment of the RAD1-RAD9-HUS1 complex onto chromatin, and in CHEK1 activation. May also serve as a sensor of DNA replication progression, and may be involved in homologous recombination.
    • 組織特異性

      Overexpressed in various cancer cell lines and in colon carcinoma (at protein level). Isoform 2 and isoform 3 are the most abundant isoforms in non irradiated cells (at protein level). Ubiquitous at low levels. Highly expressed in testis, where it is expressed within the germinal epithelium of the seminiferous tubuli. Weakly expressed in seminomas (testicular tumors).
    • 配列類似性

      Belongs to the rad17/RAD24 family.
    • 翻訳後修飾

      Phosphorylated. Phosphorylation on Ser-646 and Ser-656 is cell cycle-regulated, enhanced by genotoxic stress, and required for activation of checkpoint signaling. Phosphorylation is mediated by ATR upon UV or replication arrest, whereas it may be mediated both by ATR and ATM upon ionizing radiation. Phosphorylation on both sites is required for interaction with RAD1 but dispensable for interaction with RFC3 or RFC4.
    • 細胞内局在

      Nucleus. Phosphorylated form redistributes to discrete nuclear foci upon DNA damage.
    • Target information above from: UniProt accession O75943 The UniProt Consortium
      The Universal Protein Resource (UniProt) in 2010
      Nucleic Acids Res. 38:D142-D148 (2010) .

      Information by UniProt
    • 参照データベース

      • Entrez Gene: 5884 Human
      • Omim: 603139 Human
      • SwissProt: O75943 Human
      • Unigene: 16184 Human
      • 別名

        • CCYC antibody
        • Cell cycle checkpoint protein antibody
        • Cell cycle checkpoint protein RAD17 antibody
        • FLJ41520 antibody
        • HRAD 17 antibody
        • hRad17 antibody
        • R24L antibody
        • Rad 17 antibody
        • Rad 24 antibody
        • RAD1 (S. pombe) homolog antibody
        • RAD1 homolog antibody
        • RAD17 antibody
        • RAD17 homolog (S. pombe) antibody
        • RAD17 homolog antibody
        • Rad17 like protein antibody
        • RAD17, S. pombe, homolog of antibody
        • RAD17_HUMAN antibody
        • RAD17Sp antibody
        • Rad24 antibody
        • Rad24, mouse, homolog of antibody
        • Rad24, S. cerevisiae, homolog of antibody
        • RF C activator 1 homolog antibody
        • RF C/activator 1 homolog antibody
        • RF-C/activator 1 homolog antibody
        see all

      画像

      • Western blot - Anti-Rad17 (phospho S645) antibody (ab3620)
        Western blot - Anti-Rad17 (phospho S645) antibody (ab3620)

        Sample: Whole Cell Lysate (50µg/lane) from Hela cells treated with 10Gy IR or untreated.

        Rabbit anti-Rad 17 pSer645 (ab3620) used at 1:1,000 dilution

        Sample: Whole Cell Lysate (50µg/lane) from Hela cells treated with 10Gy IR or untreated.

        Rabbit anti-Rad 17 pSer645 (ab3620) used at 1:1,000 dilution

      プロトコール

      • Western blot protocols
      • Immunocytochemistry & immunofluorescence protocols

      Click here to view the general protocols

      データシートおよび資料

      • Datasheet
      • SDS
    • 参考文献 (4)

      ab3620 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。

      ab3620 は 4 報の論文で使用されています。

      • Alfano L  et al. Depletion of the RNA binding protein HNRNPD impairs homologous recombination by inhibiting DNA-end resection and inducing R-loop accumulation. Nucleic Acids Res N/A:N/A (2019). PubMed: 30799487
      • Shin MH  et al. ATM-dependent phosphorylation of the checkpoint clamp regulates repair pathways and maintains genomic stability. Cell Cycle 11:1796-803 (2012). PubMed: 22453082
      • Kumar A  et al. Nuclear phosphoinositide 3-kinase beta controls double-strand break DNA repair. Proc Natl Acad Sci U S A 107:7491-6 (2010). WB, ICC/IF ; Mouse . PubMed: 20368419
      • Jurvansuu J  et al. Viral transport of DNA damage that mimics a stalled replication fork. J Virol 79:569-80 (2005). PubMed: 15596849

      レビューと Q&A

      Show All レビュー Q&A
      レビューを送る 質問を送る

      1-7 of 7 Abreviews or Q&A

      Question

      Is it possible for you to send me the ab63442?
      Thanks.

      Read More

      Abcam community

      Verified customer

      Asked on Mar 07 2012

      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 1048217 (ab63442).

      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

      Abcam Scientific Support

      Answered on Mar 07 2012

      Question

      Thanks for your reply.
      I repeated the experiment with a positive and a negative control ordered from another company, and the outcome was not improved.
      So I'm wondering if you have any other antibodies for the same target recommended?
      Thanks!

      Read More

      Abcam community

      Verified customer

      Asked on Mar 06 2012

      Answer

      I am sorry to hear that the results does not improve. I can offer you another antibody we have following antibodies against the same target

      ab63442; https://www.abcam.com/Rad17-phospho-S645-antibody-ab63442.html

      ab115876; https://www.abcam.com/Rad17-phospho-S645-antibody-ab115876.html

      Please check ab115876 is a mouse polyclonal antibody, you would need anti mouse secondary if you opt for this.

      Let me know which ab you would like to try.

      Read More

      Abcam Scientific Support

      Answered on Mar 06 2012

      Question

      Thanks for your kind reply.
      I used human glioma cell line U251 in my study, and I have seen paper reported glioma cells have this pRad activity.
      I stripped and reblotted with Actin and got pretty good signal, which i guess means the transfer was good.
      I think what you said means using BSA for blocking can only reduce the background noise.
      Since I didn't even get any specific binding, I guess try again with BSA may not have signficant effect for my experiment.

      Read More

      Abcam community

      Verified customer

      Asked on Feb 22 2012

      Answer

      Well that's true. BSA reduces the background only, it does not have any effect on ab specificity.

      Anyway to proceed further in this case, I would like to ask if you are happy in trying a different lot or a different antibody. Alternatively I can also provide you full refund or credit note, which you can use for future purchases. Let me know the option you would like to go for.

      Many thanks for your cooperation in this case, I highly appreciate that.

      Read More

      Abcam Scientific Support

      Answered on Feb 22 2012

      Question

      Thanks for your reply.
      The samples that I used were whole cell lysates from cells 3 hours, 24 hours, and 48 hours post treated with 20 Gy of radiation, as well as cells without radiation as negative control.
      So I think the signal should be strong.
      By the way, what BSA solution do you suggest as blocking reagent? Do I need to use BSA solution for both primary and secondary antibodies?
      Thanks!

      Read More

      Abcam community

      Verified customer

      Asked on Feb 21 2012

      Answer

      Thank you very much for your email.

      We use 5% BSA (Sigma) in TBST for blocking and for diluting secondary and primaries. Milk in not recommended for phospho specific antibodies because milk contains casein which is a phosphoprotein; This is why it causes high background because the phospho-specific antibody detects the casein present in the milk. Please be advised that BSA will only reduce the background i.e. membrane dirtiness, it will not effect the antibody specificity. So if the membrane shows high background then I would certainly recommend trying BSA.

      The other important point is, whether the cells naturally express the protein or not. I am sorry the info of cell line wasn't included in your email so I can't be sure about it. I would however recommend using Hela lysates as positive control. Are you sing human cell line?
      Actin or GAPDH can be used as loading control. Have you tried any of these?

      I hope these suggestions will help to improve your results. If the results do not improve please do not hesitate to contact me.

      Read More

      Abcam Scientific Support

      Answered on Feb 21 2012

      Question

      It will take approximately 5 minutes to complete the questionnaire. Please fill-in all fields so that we can better assist you.

      1) Abcam product code ab3620

      2) Abcam order reference number or product batch number GR686-4

      3) Description of the problem

      No signal

      4) Sample preparation:

      Type of sample (whole cell lysates, fraction, recombinant protein…) whole cell lysates

      Lysis buffer RIPA buffer

      Protease inhibitors: Protease inhibitor cocktail

      Phosphatase inhibitors Phosphatase inhibitor cocktail

      Reducing agent SDS

      Boiling for ≥5 min? yes, 10 min

      Protein loaded ug/lane or cells/lane 51 ug

      Positive control n/a

      Negative control n/a

      5) Percentage of gel 4-20% gradient gel

      Type of membrane PVDF

      Protein transfer verified ponceau reagent

      Blocking agent and concentration 5% milk in TBST

      Blocking time 1 hour

      Blocking temperature room temperature

      6) Primary antibody (If more than one was used, describe in “additional notes”) :

      Concentration or dilution 1/500

      Diluent buffer 5% milk in TBST

      Incubation time overnight

      Incubation temperature: 4 degree


      7) Secondary antibody:

      Species:Donkey

      Reacts against: rabbit

      Concentration or dilution 1/5000

      Diluent buffer 5% milk in TBST

      Incubation time 1 hour

      Incubation temperature: room temperature

      Fluorochrome or enzyme conjugate:HRP

      8) Washing after primary and secondary antibodies:

      Buffer TBST

      Number of washes 3 times, 5 minutes each

      9) Detection method Chemiluminascence

      10) How many times have you run this staining? twice

      Do you obtain the same results every time? yes

      What steps have you altered to try and optimize the use of this antibody? no

      Read More

      Abcam community

      Verified customer

      Asked on Feb 20 2012

      Answer

      Thank you for your enquiry regarding ab3620 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

      The protocol looks fine to me however I would like to make few comments/suggestions that might help to improve your results and that you could consider trying:

      ab3620 is a phospho specific antibody. We used 10Gy IR treated Hela cells to test the specificity of this antibody. I would recommend treating your sample cells there by inducing the phosphorylatuion of amino acid chemically or by radiations.

      Milk is not recommended as blocking reagent with phospho specific antibodies. Please use BSA.

      Please try both of these suggestions and let us know the results; we will be happy to help further.

      Read More

      Abcam Scientific Support

      Answered on Feb 20 2012

      Question

      Hi,
      I ordered an anti-pRad17 antibody (Ab3620) recently and tested on my cell lysates for western blot.
      I started with cells with and without radiation and expected an enhanced pRad17 activity in cells with radiation.
      However, I didn't get any signal from my blot.
      I'm wondering if you can help me troubleshooting with my western blot experiment?
      Thanks!

      Read More

      Abcam community

      Verified customer

      Asked on Jan 23 2012

      Answer

      I am sorry to hear that you have been experiencing problems using this product in the application that you wish.



      In order to assess the quality of our products I would ask that you complete a brief questionnaire relating to the application used. Often it is possible to make suggestions that may help resolve problems experienced using a particular product.



      As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.



      All our customer feedback, including complaints are monitored weekly by our in house technical support team. If a product is at fault the technical support team will consider removing the product from our catalogue in order to avoid future customer inconvenience.



      Please could you provide some further details of the protocol used and complete the following form (attached as a word document). It would be much appreciated if you could attach an image to the response.



      Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.

      Read More

      Abcam Scientific Support

      Answered on Jan 23 2012

      Question

      I am interested in your human rad 17 antibodies and was concerned that your goat polyclonal recognises a 90 kD protein, whereas your rabbit polyclonal recognises a 69kD. The phospho specific ( and therefore I assume slower running) comes in at 85-88kD. Is there a reason that your polyclonal goat recognises larger protein?

      Read More

      Abcam community

      Verified customer

      Asked on May 18 2003

      Answer

      To our knowledge, these three antibodies have not been used in parallel, which would be required to determine if there are truely differences in the apparant molecular weight of the protein recognized by each antibody. The differences in the reported apparant molecular weights of the proteins detected by the 3 antibodies is likely owing to differences in molecular weight markers that where used, position of the sample relative to the positon of the molecular weight markers in the gel, and size and composition of the resolving gels. The difference in relative mobility of the protein recognized by the goat anti-Rad17 and the phospho specific antibody is certainly within experimental error.

      Read More

      Abcam Scientific Support

      Answered on May 20 2003

      Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
      For licensing inquiries, please contact partnerships@abcam.com

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