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RabMAb

Anti-Catalase 抗体 [EP1929Y] - Peroxisome Marker (ab76024)

製品の概要

  • 製品名
    Anti-Catalase antibody [EP1929Y] - Peroxisome Marker
    Catalase 一次抗体 製品一覧
  • 製品の詳細
    Rabbit monoclonal [EP1929Y] to Catalase - Peroxisome Marker
  • 由来種
    Rabbit
  • アプリケーション
    適用あり: WB, ICC, IHC-Pmore details
    適用なし: Flow Cyt or IP
  • 種交差性
    交差種: Human
  • 免疫原

    Synthetic peptide within Human Catalase aa 400-500 (C terminal). The exact sequence is proprietary.
    Database link: P04040
    (Peptide available as ab225865)

  • ポジティブ・コントロール
    • WB: HeLa cell lysate. IHC-P: human brain tissue.
  • 特記事項

    Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab76024 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 1/10000 - 1/20000. Detects a band of approximately 60 kDa (predicted molecular weight: 60 kDa).Can be blocked with Human Catalase peptide (ab225865).
ICC 1/100 - 1/250.
IHC-P 1/100 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
  • 追加情報
    Is unsuitable for Flow Cyt or IP.
  • ターゲット情報

    • 機能
      Occurs in almost all aerobically respiring organisms and serves to protect cells from the toxic effects of hydrogen peroxide. Promotes growth of cells including T-cells, B-cells, myeloid leukemia cells, melanoma cells, mastocytoma cells and normal and transformed fibroblast cells.
    • 関連疾患
      Defects in CAT are the cause of acatalasia (ACATLAS) [MIM:115500]; also known as acatalasemia. This disease is characterized by absence of catalase activity in red cells and is often associated with ulcerating oral lesions.
    • 配列類似性
      Belongs to the catalase family.
    • 翻訳後修飾
      The N-terminus is blocked.
    • 細胞内局在
      Peroxisome.
    • Information by UniProt
    • 参照データベース
    • 別名
      • Cas1 antibody
      • CAT antibody
      • CATA_HUMAN antibody
      • Catalase antibody
      • Cs1 antibody
      • MGC138422 antibody
      • MGC138424 antibody
      see all

    画像

    • Lane 1: Wild type HAP1 whole cell lysate (20 µg)
      Lane 2: CAT knockout HAP1 whole cell lysate (20 µg)
      Lane 3: HeLa whole cell lysate (20 µg)

      Lanes 1 - 3: Merged signal (red and green). Green - ab76024 observed at 60 kDa. Red - loading control, ab9484, observed at 37 kDa.

      ab76024 was shown to specifically react with CAT when CAT knockout samples were used. Wild-type and CAT knockout samples were subjected to SDS-PAGE. Ab76024 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 5000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

    • Anti-Catalase antibody [EP1929Y] - Peroxisome Marker (ab76024) at 1/20000 dilution + HeLa cell lysate at 10 µg

      Secondary
      HRP labelled goat anti-rabbit at 1/2000 dilution

      Predicted band size: 60 kDa
      Observed band size: 60 kDa

    • Immunohistochemical staining of Catalase in paraffin embedded human normal brain tissue using ab76024 at a 1/100 dilution.

    参考文献

    This product has been referenced in:
    • Cai M  et al. Disruption of peroxisome function leads to metabolic stress, mTOR inhibition, and lethality in liver cancer cells. Cancer Lett 421:82-93 (2018). ICC/IF . Read more (PubMed: 29458144) »
    • Kampen KR  et al. The ribosomal RPL10 R98S mutation drives IRES-dependent BCL-2 translation in T-ALL. Leukemia N/A:N/A (2018). Read more (PubMed: 29930300) »
    See all 8 Publications for this product

    レビューと Q&A

    1-7 of 7 Abreviews or Q&A

    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 948366. 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
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (A549 lung cancer cells)
    Gel Running Conditions
    Reduced Denaturing (4-20% Tris-glycine)
    Loading amount
    15 µg
    Specification
    A549 lung cancer cells
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C

    Abcam user community

    Verified customer

    投稿 Apr 21 2015

    Answer

    Thank you for contacting us.

    I would like to apologize for this error. When we received the information, that the antibody has been tested in the laboratory with negative results on rat tissue, we updated immediately the datasheet. This was last automn. Indeed, I do not know why we have not sent out the emails to the customers to inform about the issue. I would double check this with the responsable people. Thank you.

    I am very sorry that the customerhad problems with this antibody. Please remind the customer, that if in the future he/she has some problems with any of our product, to contact us as soon as possible. Our technical support team is very happy to help to troubleshoot and improve results. In the case an antibody is not working as described, we are happy to replace it (in accordance with our Abpromise guarantee).

    Please confirm the order number (or approximate date) of the order of this antibody and I will arrange for the credit. If instead of a credit notethe customerwould prefer an alternative antibody as recompensation, please do let me know.

    Thank you for your cooperation and support in this case. I am looking forward to hear back from you onhow you would like to proceed.

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    Answer

    The ab16731 is not specific to N-terminal end of the protein. It is produced using recombinant fragment of whole protein and because it is a polyclonal antibody so it will bind to multiple epitopes. It is likely that ab16731 will be able to detect the catalase in your sample. However we can not 100% confirm this without comparison with positive control. I am sending you the lysates ab29545 (Hela cell lysates) which you can use as positive control. I would also recommend trying ab98210 side by side which will help to determine whether the lysates were problematic or antibodies. The Order number I have created for these products is 975321. You will be receiving these products on Monday. I will look forward to receiving the results soon.  

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    Answer

    Thank you for sending the image which is very self explanatory for multiple bands. Please ignore my suggestions for multiple bands as these were more general rather than related to this case. The antibody (ab98210) I recommended was the second most suitable antibody based on your immunogen requirements. I am sorry that the antibody did not give expected results. In order to help further I have compiled an excel file of all the anti-catalase antibodies we have in catalogue, along with their immunogen details. Could you please have a look and choose the antibody you would like to try. Unfortunately the immunogen of all these antibodies is from human Catalase protein which is not 100% homologues to Rat protein or at least in the sequence requirement as per your mass spec data. I therefore would be happy to refund the money in case you could not find the product which is a perfect match. Please click to open the sequence similarities info between Rat and Human protein; http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolresult.ebi?tool=clustalw2&jobId=clustalw2-I20111027-112818-0636-62047582-pg Please choose product from the attached list and contact me for replacement. I would again suggest using human lysates as a positive control. I hope these suggestions will be helpful. Should you have question please do not hesitate to ask.  

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    Answer

    Thank you for your email. I am sorry to hear that the results did not improve. The multiple band problem could be due to following reasons: - High protein conc. of lysates per well - High conc. of primary and secondary ab - Cell confluence or cell passage - Cleavage products -  Active protease inhibitors etc.  I would like also to ask about the step you have altered for troubleshooting purpose. Could you send me an image and lysates details? Have you used any positive control lysates also? Looking forward to hearing from you soon.

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    Question

    1) Abcam product code ab76024 2) Abcam order reference number or product batch number lot:GR1737-3 3) Description of the problem The antibody is not detecting any catalase in my samples that have previously shown a large amount of catalase by mass spectrometry.See attached pptx file which in yellow highlights all peptides detected by mass spectrometry on same sample. Catalase is in my sample based on many spectra and peptides identified for this protein. 4) Sample preparation: Type of sample: Proximal convoluted cells isolated from rat kidneys and organelle(mitochondria, peroxisomes, & etc) isolated by sucrose gradient Lysis buffer: RIPA Protease inhibitors: PMSF Phosphatase inhibitors; NONE Reducing agent: BME Boiling for ≥5 min? Yes and no, I have tired both ways Protein loaded ug/lane or cells/lane Positive control: Whole cell lysate from Proximal convoluted cells isolated from rat kidneys which little is detected. Negative control 5) Percentage of gel: 10% Type of membrane: PVDF Protein transfer verified: YES Blocking agent and concentration: Thermo Non-protein blocking buffer Blocking time: 2 hours Blocking temperature: RT 6) Primary antibody (If more than one was used, describe in “additional notes”) : Concentration or dilution Diluent buffer: Blocking buffer with 0.1% Tween Incubation time: O/N Incubation temperature:4 degrees 7) Secondary antibody: Species: mouse Reacts against: Rabbit Concentration or dilution: 1:10,000 Diluent buffer Blocking buffer with 0.1% Tween & 0.01% SDS Incubation time: 30 minutes Incubation temperature: RT Fluorochrome or enzyme conjugate:Fluorochrome 8) Washing after primary and secondary antibodies: Buffer; TBS 0.1% Tween Number of washes 3X 9)Detection method 10) How many times have you run this staining? All the time nothing new in our lab. Do you obtain the same results every time? What steps have you altered to try and optimize the use of this antibody? The following dilutions of primary tired: 1/10,000, 1/5,000, 1/2,000, 1/1,000 all O/N 4degrees See attached file and let me know if you need more details.

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    Answer

    Thank you for contacting Abcam Technical Team 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 product. I have been discussing this issue with some colleagues in the Lab. Regrettably, I have been informed that ab76024 is no longer recommended for use in rat samples based on internal test results. I have just amended the on-line datasheet to reflect this. I appreciate the time you have spent on these experiments in the laboratory and it is disappointing the results have not been successful. I am very sorry for this discrepancy and can certainly offer you either an immediate refund or a credit note which you can use in the future. Please do let me know how you wish to proceed with your enquiry.

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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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