Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Catalase antibody [EP1929Y] - Peroxisome Marker (ab76024)

Reviews (1)Q&A (6)References (12)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EP1929Y] to Catalase - Peroxisome Marker
  • Suitable for: ICC/IF, WB, IHC-P
  • Knockout validated
  • Reacts with: Human

Overview

  • Product name

    Anti-Catalase antibody [EP1929Y] - Peroxisome Marker
    See all Catalase primary antibodies

  • Description

    Rabbit monoclonal [EP1929Y] to Catalase - Peroxisome Marker

  • Host species

    Rabbit

  • Tested applications

    Suitable for: ICC/IF, WB, IHC-Pmore details
    Unsuitable for: Flow Cyt or IP

  • Species reactivity

    Reacts with: Human

  • Immunogen

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

  • Positive control

    • WB: HeLa cell lysate. IHC-P: human brain tissue, human bladder cancer tissue. ICC/IF: HeLa cells.

  • General notes

    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.

Properties

Applications

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.

Application Abreviews Notes
ICC/IF 1/100 - 1/250.
WB 1/10000 - 1/20000. Detects a band of approximately 60 kDa (predicted molecular weight: 60 kDa).
IHC-P 1/100 - 1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
  • Application notes
    Is unsuitable for Flow Cyt or IP.

    • Target

    • Function

      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.

    • Involvement in disease

      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.

    • Sequence similarities

      Belongs to the catalase family.

    • Post-translational
      modifications

      The N-terminus is blocked.

    • Cellular localization

      Peroxisome.
    • Information by UniProt

    • Database links

      • Alternative names

        • Cas1 antibody
        • CAT antibody
        • CATA_HUMAN antibody
        • Catalase antibody
        • Cs1 antibody
        • MGC138422 antibody
        • MGC138424 antibody
        see all

      Images

      • Western blot - Anti-Catalase antibody [EP1929Y] - Peroxisome Marker (ab76024)
        Western blot - Anti-Catalase antibody [EP1929Y] - Peroxisome Marker (ab76024)
        All lanes : Anti-Catalase antibody [EP1929Y] - Peroxisome Marker (ab76024) at 1/10000 dilution

        Lane 1 : Wild-type HeLa cell lysate
        Lane 2 : CAT knockout HeLa cell lysate

        Lysates/proteins at 20 µg per lane.

        Performed under reducing conditions.

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



        Lanes 1-2: Merged signal (red and green). Green - ab76024 observed at 60 kDa. Red - loading control ab8245 observed at 37 kDa.

         ab76024 Anti-Catalase antibody [EP1929Y] - Peroxisome Marker was shown to specifically react with Catalase in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265250 (knockout cell lysate ab256859) was used. Wild-type and Catalase knockout samples were subjected to SDS-PAGE. ab76024 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

         

         

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Catalase antibody [EP1929Y] - Peroxisome Marker (ab76024)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Catalase antibody [EP1929Y] - Peroxisome Marker (ab76024)

        Immunohistochemical analysis of Paraffin-embedded human bladder cancer tissue sections labeling Catalase with ab76024 at 1/1000. Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Sections were counterstained with Hematoxylin. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0).

        Granular cytoplasmic staining on human bladder cancer.

      • Western blot - Anti-Catalase antibody [EP1929Y] - Peroxisome Marker (ab76024)
        Western blot - Anti-Catalase antibody [EP1929Y] - Peroxisome Marker (ab76024)
        All lanes : Anti-Catalase antibody [EP1929Y] - Peroxisome Marker (ab76024)

        Lane 1 : Wild-type HAP1 whole cell lysate
        Lane 2 : CAT knockout HAP1 whole cell lysate
        Lane 3 : HeLa whole cell lysate

        Lysates/proteins at 20 µg per lane.

        Predicted band size: 60 kDa



        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 1/5000 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.

      • Immunocytochemistry/ Immunofluorescence - Anti-Catalase antibody [EP1929Y] - Peroxisome Marker (ab76024)
        Immunocytochemistry/ Immunofluorescence - Anti-Catalase antibody [EP1929Y] - Peroxisome Marker (ab76024)

        Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma epithelial cell) labeling Catalase ab76024 at 1/100. Cells were fixed with 100% Methanol. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. 

        ab195889, Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/100 was used as counterstain antibody.

        Confocal image showing membranous staining in HeLa cells.

         

      • Western blot - Anti-Catalase antibody [EP1929Y] - Peroxisome Marker (ab76024)
        Western blot - Anti-Catalase antibody [EP1929Y] - Peroxisome Marker (ab76024)
        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

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Catalase antibody [EP1929Y] - Peroxisome Marker (ab76024)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Catalase antibody [EP1929Y] - Peroxisome Marker (ab76024)

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

        Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

      References (12)

      Publishing research using ab76024? Please let us know so that we can cite the reference in this datasheet.

      ab76024 has been referenced in 12 publications.

      • Wang T  et al. Synergistic antitumour effects of triptolide plus 10-hydroxycamptothecin onbladder cancer. Biomed Pharmacother 115:108899 (2019). PubMed: 31063955
      • 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 . 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). PubMed: 29930300
      • Irías-Mata A  et al. a-Tocopherol transfer protein does not regulate the cellular uptake and intracellular distribution of a- and ?-tocopherols and -tocotrienols in cultured liver cells. Redox Biol 19:28-36 (2018). PubMed: 30098456
      • Hu Q  et al. Knockdown of SIRT1 Suppresses Bladder Cancer Cell Proliferation and Migration and Induces Cell Cycle Arrest and Antioxidant Response through FOXO3a-Mediated Pathways. Biomed Res Int 2017:3781904 (2017). PubMed: 29147649
      View all Publications for this product

      Customer reviews and Q&As

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      1-7 of 7 Abreviews or Q&A

      Question

      Can you please send me the ab98210 and the ab25987 ASAP. I spent a considerable amount of time and sample on the previous antibody and I need to get these westerns done so I can publish. I noticed the ab25987 has a lead time of 4-6wks so please send the ab98210 right away since the lead time is 1-2 wks and I need to get going on these westerns. I am happy to share any western data with my rat samples to update the datasheets for you.

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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 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.

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      Western blot abreview for Anti-Catalase antibody [EP1929Y] - Peroxisome Marker

       Excellent
      Abreviews
      abreview image
      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
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      Abcam user community

      Verified customer

      Submitted Apr 21 2015

      Question

      Greetings-

      I would like to inform you aboutthe customer complaint.
      Some time ago my clientordered antibody ab76024, which was dedicated to work on rats.
      The client was not happy with the results of experiments. The results were the main topic of her research.
      After some time, she decided to return to studies using this antibody. The client is very surprised by the fact that Abcam deleted the informationthat this antibody is suitable for use in the rat.
      The client feels cheated andexpects adequate compensation for financial losses.
      I wouldlike to mentionthat we have not received any indication that this antibody has changed its characteristics of which I should informmy customers.

      I look forward to favorable consideration of this complaint.


      Yours faithfully,

      Aleksandra Kulma
      Sales Specialist - Genetic Identity



      Symbios Sp. z o.o.

      tel./fax: +48 58692 80 83

      http://www.symbios.com.pl/
      ul. Modrzewiowa 37
      83-010 Straszyn
      NIP: 957 07 03157

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      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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      Question

      I looked over your antibody list. Thank you for providing that. Based on my mass spec peptide coverage and the rat to human homology it looks like N-term raised antibodies are the best. I found ab16731 to look the most promising there is even a western on the website of rat liver tissue with a very clean one band! I am not sure to which antibody on your excel sheet this matches to but I think it is #27. Anyways if you could send ab16731 ASAP, if you think it is a good one for me to try? There are so many antibodies to try for catalase but this had a lot of different tissue types from rat that it has worked in so hopefully it will work in kidney. Also I have no human lysates to use for a control could you send me some? Thank you for all your help.

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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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      Question

      I loaded 10 micrograms of my subcellular fraction. I had tested it on a whole cell lysate before and determined 1:000 dilution was optimal to get any band to appear at 60kDa but still had brighter upper bands. I disagree with your reasoning. It can't not be cleavage products because all the extra bands are a larger weight. I used my secondary very dilute 1:10,000. Cell confluence or passage is irrelevant because the samples are from rat kidney tissue. The image is attached of my subcellular fractions which I got from rat kidney cortex tissue and isolated a subcellualr fraction from a sucrose density gradient. I can't do trouble shooting with abcam antibodies anymore the last catalase antibody I troubleshooting so much I waste a lot of sample and it wasn't my problem it was abcam that said it reacted to rat when it didn't. I have no positive control. I would really like help on this problem or another antibody that will work. Thank you

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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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      Question

      My subcellular fraction which was the samples I identified catalase by mass spec in is not showing a specific 60kDa band. What I am detecting is many bands larger than 60kDa which makes believe the antibody is not working(see attached). Please offer an advice you have or should I try a different antibody.Thanks

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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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