Anti-Citrulline antibody (ab100932)

Reviews (2)Q&A (16)References (10)
ELISA - Anti-Citrulline antibody (ab100932)
  • ELISA - Anti-Citrulline antibody (ab100932)
  • Immunocytochemistry - Anti-Citrulline antibody (ab100932)

Key features and details

  • Rabbit polyclonal to Citrulline
  • Suitable for: ELISA, ICC
  • Reacts with: Species independent
  • Isotype: IgG

Overview

  • Product name

    Anti-Citrulline antibody
    See all Citrulline primary antibodies

  • Description

    Rabbit polyclonal to Citrulline

  • Host species

    Rabbit

  • Specificity

    ab100932 reacts specifically with intrapeptidic citrulline independently of the amino acid sequence. It does not react with free citrulline, ornithine or arginine. Citrulline is an un-natural amino-acid and its expression is very low. Factors limiting the detection are the amount of endogenous citrulline within a protein and the accessibility of the antibody to these sites.

  • Tested applications

    Suitable for: ELISA, ICCmore details
    Unsuitable for: WB

  • Species reactivity

    Reacts with: Species independent

  • Immunogen

    Citrulline coupled to KLH via Glutaraldehyde

  • Positive control

    • Hypercitrullinated Histones induced in HL60 cells by DMSO +calcium +calcium ionophore A23187

  • General notes

    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.

Properties

Applications

Our Abpromise guarantee covers the use of ab100932 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
ELISA Use at an assay dependent concentration.
ICC Use at an assay dependent concentration.
  • Application notes
    Is unsuitable for WB.

    • Target

    • Relevance

      The amino acid Citrulline is required to detoxify the liver from ammonia, which is a waste product of the body from oxidation. Citrulline promotes energy and assists with the immune system. This unusual amino acid is formed in the urea cycle by the addition of carbon dioxide and ammonia to ornithine. It is then combined with aspartic acid to form arginosuccinic acid, which later is metabolized into the amino acid arginine.

    Images

    • ELISA - Anti-Citrulline antibody (ab100932)
      ELISA - Anti-Citrulline antibody (ab100932)

      ELISA data showing the high immuno-reactivity of ab100932 against citrulline due to the high amounts of citrulline conjugated to BSA.

    • ELISA - Anti-Citrulline antibody (ab100932)
      ELISA - Anti-Citrulline antibody (ab100932)

      ELISA data demonstrating the specificity of ab100932 to citrulline naturally present in histones (endogenous citrulline).

    • Immunocytochemistry - Anti-Citrulline antibody (ab100932)
      Immunocytochemistry - Anti-Citrulline antibody (ab100932)This image is courtesy of an anonymous Abreview
      Immunocytochemical analysis of Human buccal mucosal cells, staining Citrulline with ab100932.

      Cells were fixed with acetone followed by 2% glutaraldehyde, permeabilized with 0.1% Triton X-100 and blocked with 3% BSA for 20 minutes at 22°C. Samples were incubated with primary antibody (1/200 in PBS) for 15 hours at 4°C. An undiluted biotinylated anti-rabbit polyclonal IgG was used as the secondary antibody.

      See Abreview

    References (10)

    Publishing research using ab100932? Please let us know so that we can cite the reference in this datasheet.

    ab100932 has been referenced in 10 publications.

    • Fonseca Z  et al. Pathogenic Entamoeba histolytica, but not Entamoeba dispar, induce neutrophil extracellular trap (NET) formation. J Leukoc Biol 105:1167-1181 (2019). PubMed: 30913315
    • Verheul MK  et al. Pitfalls in the detection of citrullination and carbamylation. Autoimmun Rev 17:136-141 (2018). PubMed: 29203292
    • Hutchinson D  et al. Cadmium nanoparticles citrullinate cytokeratins within lung epithelial cells: cadmium as a potential cause of citrullination in chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis 13:441-449 (2018). PubMed: 29430177
    • Burbano C  et al. Potential Involvement of Platelet-Derived Microparticles and Microparticles Forming Immune Complexes during Monocyte Activation in Patients with Systemic Lupus Erythematosus. Front Immunol 9:322 (2018). PubMed: 29545790
    • Fonseca Z  et al. Entamoeba histolytica Induce Signaling via Raf/MEK/ERK for Neutrophil Extracellular Trap (NET) Formation. Front Cell Infect Microbiol 8:226 (2018). PubMed: 30023352
    • Villar-Vesga J  et al. Platelet-derived microparticles generated in vitro resemble circulating vesicles of patients with rheumatoid arthritis and activate monocytes. Cell Immunol N/A:N/A (2018). PubMed: 30538031
    • Burbano C  et al. Extracellular vesicles are associated with the systemic inflammation of patients with seropositive rheumatoid arthritis. Sci Rep 8:17917 (2018). PubMed: 30559453
    • Hutchinson D  et al. Carbamylation/citrullination of IgG Fc in bronchiectasis, established RA with bronchiectasis and RA smokers: a potential risk factor for disease. ERJ Open Res 3:N/A (2017). PubMed: 29204430
    • Yamakawa M  et al. Porphyromonas gingivalis infection exacerbates the onset of rheumatoid arthritis in SKG mice. Clin Exp Immunol 186:177-189 (2016). PubMed: 27465496
    • Deplus R  et al. Citrullination of DNMT3A by PADI4 regulates its stability and controls DNA methylation. Nucleic Acids Res N/A:N/A (2014). WB . PubMed: 24957603

    Customer reviews and Q&As

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    11-18 of 18 Abreviews or Q&A

    Question

    Hello,
    Okay then we will try the ab6464. I am not the person who submit the order so I will find out about this. Does it make a difference if it is ordered through a subdealer? It is still a Abcam ab. Please let me know about this.
    Best Regards

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    Answer

    Thank you for your reply.

    The Abpromise guarantee is only valid for products bought direct from Abcam or one of our authorized distributors. The terms and conditions of the guarantee can be found on https://www.abcam.com/abpromise.

    I look forward to your reply with the order details.

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    Question

    Hello,
    Thanks for the reply. We cant find other explanation than the product does not work eigther.
    Would it be possible to send a different lot number of ab100932 and the ab6464 ? We can provide you results in regard to ELISA with both abs and WB in near future.
    Els we will try the ab6464 instead of since other have positive results in other aplications than ELISA with this ab.

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    Answer

    Thank you for your reply.

    As a compensation I can send to you one unit of ab6464, unfortunately I cannot send both ab6464 and ab100932.

    There is no order in our system corresponding to the address you provided in your last email. Could you please send me an order number as well as the date the order was placed? Have you ordered the product directly at Abcam orvia a subdealer?

    I look forward to your reply.

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    Question

    Inquiry: I could see another costumer had problems with same product - submitted same day our product was bought.
    There might be a general problem?? This is an indirect ELISA on proteins with/without citrullines
    We have coated plates with 1 ug/mL fibrinogen (citrullinated and not citrullinated. This citrullation was confirmed otherwise as well) on 96-wells microplate (carbonatebuffer O/N at 4 degrees) (also a non-protein containing).
    Plates were washed 4 times and blocked in PBS tween20 0.05% for 30 min at RT.
    ab100932 was diluted from 1:100 and added for incubation in PBS tween20 0.05% for 2 hours at RT
    Again washed 4 times - incubated with Peroxidase conjugated Goat polyclonal Anti-rabbit IgG (dako) 1 hour at RT in same buffer at RT.
    Again washed 4 times followed by OPD substrabe developement.
    As attached dato shows - there is no difference between reaction against fibrinogen and fibrinogen containing citrulline usin ab100932.
    The small difference at dilution 1:100 is not satisfactory and at 1:400 we got a higher signal on fibrinogen than fibrinogen containing citrullines.
    We do a lot of ELISA and all buffers, secondary reagents are used for lots of assays in our laboratory.
    Let me know if you can help me out.
    Best Regards

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    Answer

    Thank you for contacting us. I am sorry to hear this antibody is 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. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.

    Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial.

    I appreciate the time you have spent on these experiments and would be pleased to arrange in compensation a refund,a credit note ora replacement with a different lot number of ab100932 or an alternative anti-citrulline such as ab6464 (https://www.abcam.com/ab6464).

    Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.
    Could you also please send me one of the following : the order number, the PO number, or the address of your lab?

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    Question

    Inquiry: Antibody Storage Conditions (temperature/reconstitution etc) Aliquot and store at -80C Description of the Problem (high background, no signal, non-specific colour development, etc) No signal development on tested samples and positive control colour development on negative controls. Type of ELISA (Direct ELISA/Indirect ELISA/Sandwich ELISA etc.) Indirect ELISA Sample (Species/Tissue/Cell Type/Cell Line etc) Coating well (Buffer/Concentration of the coating material etc.) - 96-wells microplate in CCP3 ELISA kit purchased from INOVA Diagnostic Inc - Manually coated synthetic peptides: incubate with bicarbonate buffer pH 9.6 at 37C for 2 hours. Concentration of peptides is 10 ug/ml. Blocking Conditions (Buffer/time period, Blocking agent etc.) 1% BSA in PBS Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, wash step) Rabbit polyclonal Anti-citrulline IgG (ab100932) Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, wash step) Peroxidase conjugated Goat polyclonal Anti-rabbit IgG Diluent: HRP stabilizer Incubation: 37C for one hour Washing: 4 times with PBS containing Tween20 Detection Method (Substrate/Diluent etc) TMB Positive and negative controls used (please specify) Positive Control: 96-wells microtiter plate in CCP3 ELISA kit purchased from INOVA Diagnostic Inc Synthesized citrullinated JO1, SmB, SmD, TOPO1 peptides using the FMOC-chemistry, the peptide sequence were tested by liquid chromatography (HPLC) and mass spectrometry by xx. Negative Control: Synthesized Double-strand DNA and JO1, SmB, SmD, TOPO1 peptides which do not containing any citrulline OPTIMIZATION ATTEMPTS (PROBLEM SOLVING) How many times have you tried the antibodies? 6 times (by two independent scientists) Have you run a “No Primary” control? Yes/No (Delete one) Yes Do you obtain the same results every time? Yes/No (Delete one) Yes What steps have you altered? Three different types of functional secondary antibodies systems have been used so far, but it still didn’t work out.

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    Answer

    Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear this antibody is not providing satisfactory results.

    The details provide us with vital information for monitoring product quality. Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial. I am very sorry for the customer. I apologize for the inconvenience and am pleased to offera replacement antibody or acredit note if the antibody has been purchased in the last months, according to our guarantee. Please provide us with the order number of this antibody that we can identify the vial as well as apply the guarantee.

    Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

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    Question

    Hi tech support, how are you?

    Please advise on the difference(s) in terms of advantage(s) between the two items below:

    AB100932 and

    AB6464

    Thank you so much!

    Kind regards,

    Read More
    Answer

    Thank you for your inquiry! We are well, thanks- Ihope you are well too!

    The two products are from two different laboratories and therefore productions. They are both against bound citrulline and guaranteed by us in ICC/IF, ELISA, IHC-Fr for the ab6464 and ELISA, ICC, WB for the ab100932. If the customer wants to do Western blot, I can recommend the ab100932. If the customer wants to do frozen section immunohistochemistry, I recommend the ab6464.

    Other differences are the information or the unit size. The ab100932 comes in 200ul and for the ab6464 we have the crossreactivity data against homocitrulline for example on the datasheet.

    I hope this information is helpful. Please do not hesitate to contact us again should you have any other question.

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    Question


    Thank you very much for your detailed answers. I think I will choose ab100932 and the ab97092.
    Thanks a lot for the fast and comprehensive reply.

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    Answer

    Köszönöm for your message!

    Just let me know if there is anything else I can help you with.

    Have a nice weekend!

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    Question

    I would like to do an anti-citrulline ELISA to quantify citrullinated proteins. I would like to choose the ab6464 anti citrulline polyclonal primary and ab97092 secondary antibody. My questions are: 1. The primary antibody was raised against glutaraldehyde congjugated citrulline – would that recognize the citrullins on the proteins? 2. Do I have to use BSA-glutaraldehyde in Ab dilution serum? 3. What is the main difference between ab6464 and ab100932? Which one would you recommend for use for citrullinated protein detection? Thanks in advance for yor answers!

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    Answer

    Thank you for contacting us.

    Regarding your questions I am happy to let you know the following:

    1) Indeed, the Anti-Citrulline antibodyab6464 would recognise citrullins on proteins. In fact it would recognise citrullins only on proteins and not free citrulline.

    2) There is no need to use BSA-glutaraldehyde in dilution buffers.

    3) I suppose the main difference between ab6464 and ab100932 is the characterisation of these antibodies:

    ab6464 has been tested and is guaranteed to work in ELISA, immunocytochemistry/immunofluorescence (ICC/IF) and immunohistochemistry on frozen sections (IHC-Fr), whereas ab100932 works in ELISA, immunocytochemistry/immunofluorescence (ICC/IF) and Western blot.

    Also, ab6464 has been shown to detect only citrulline residues on proteins not free citrulline. ab100932 has also beenshownnot to react with free citrulline, ornithine or arginine.

    ab100932 reacts specifically with intrapeptidic citrulline independently of the amino acid sequence, if this is of importance for your experimental setting.

    Another (though minor) difference is the immunogen: For ab6464 the small molecule is coupled via glutaraldehyde to BSA, for ab100932 it is conjugated to KLH. This, however, would only affect the immunogenicity elicited in the rabbits; it has no effect on the reactivity of these antibodies.

    Taken all these factors together, the choice of the primary antibody is yours. If you have for example a future Western blot application in mind, I would recommend ab100932, for a potential IHC-Fr experiment I would suggest ab6464.

    4) Just a thought to the choice of secondary antibody: ab97092 is suitable for either ab6464 or ab100932, if you prefer to use the Alkaline Phosphatase (AP) conjugate. We also have suitable HRP-conjugated secondary antibodies available, such as ab97095 or ab97051.For this decision, it is important to consider the fact that some biological materials have high levels of endogenous enzyme activity (such as high AP in alveolar cells, high peroxidase in red blood cells) and this may result in non-specific signal, as I am sure you are aware of. If necessary, perform an additional blocking treatment with levamisol (for AP) or with 0.3% solution of H2O2 in methanol (for peroxidase).

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

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    Question

    LOT NUMBER GR13102-3 ORDER NUMBER 3121989 DESCRIPTION OF THE PROBLEM Does not pick up positive control and multiple bands detected from total cell lysate cannot be verified as citrullinated. SAMPLE HCT116 cell lysate and Keratine from human epidermis (Sigma K0253) as positive control PRIMARY ANTIBODY Abcam ab100932 in buffer described above 1:500 no signal/weak signal 1:250 unspecific signal 1-2 h incubation @ RT 3x10min wash in buffer DETECTION METHOD Supersignal west dura extended duration substrate (Thermo Scientific) Tried detection on both film and digital developper POSITIVE AND NEGATIVE CONTROLS USED Positive control Keratine from human epidermis (see above) ANTIBODY STORAGE CONDITIONS Aliquoted upon arrival and stored at -20oC SAMPLE PREPARATION Standard lysate buffer containing CompleteMINI inhibitor (Roche) AMOUNT OF PROTEIN LOADED 30 ug totoal cell lysate and 9 ug keratin ELECTROPHORESIS/GEL CONDITIONS Reducing gels 4-12%. Everything from Invitrogens NuPAGE system TRANSFER AND BLOCKING CONDITIONS Transfer to PVDF with Invitrogen XCell. Blocking, 1st and 2nd antibody in 5% BSA in TBST or PBST buffer (both tried) SECONDARY ANTIBODY DAKO Polyclonal goat anti rabbit -HRP in buffers described above 1:5000 1 h incubation @ RT 3x10 min wash in buffer HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No WHAT STEPS HAVE YOU ALTERED? Dilution of primary antibody, strenght and composition of buffer (TBST and PBST), detection method (film or digital developper) ADDITIONAL NOTES No indications for dilution of antibody given in datasheet from abcam, and no picture of performance and no suggestion for positive control. 1:500 dilution hardly gives a signal, and does not pick up positive control (suggested by Millipore). Multiple bands detected in total cell lysate, but not possible to verify if these are specific or not. 1:250 dilution gives unacceptable high bacground. Please advise on positive control or proof of specificity for this antibody.

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    Answer

    Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results and I apologize for the delay as I was awaiting more information from the testing laboratory. The lab did not have a positive control testing WB image, however, they were able to pass along the protocol tested: The positive control was HL60 cells treated with DMSO or with DMSO and A23187. The histones of these cells were then extracted. The sample were loaded on a 14% acrylamide SDS PAGE gel. The transfer was made on a PVDF membrane The blocking buffer was PBS 1X with 5% BSA The antibody was used at 1/1000 in a TBST Tween 20 buffer with 1% BSA. The antibody was incubated overnight at +4°C. Would you be able to run a positive control with treated HL60 cells? Also incubating the primary antibody overnight at 4C may help. This is our suggested histone extraction protocol: https://www.abcam.com/index.html?pageconfig=resource&rid=11410 I hope these suggestions improve your results. Please let me know if they help. If not, we would be happy to replace or refund the antibody for you in accordance with our Abpromise.

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    11-18 of 18 Abreviews or Q&A

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