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    citrulline-antibody-ab6464.pdf

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Signal Transduction Metabolism Amino Acids
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Anti-Citrulline 抗体 (ab6464)

  • Datasheet
  • SDS
Reviews (6)Q&A (18)References (13)

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Abpromise

保証された製品品質、優れたカスタマー・サポート。 詳細はこちら。

Immunocytochemistry/ Immunofluorescence - Anti-Citrulline antibody (ab6464)

    Key features and details

    • Rabbit polyclonal to Citrulline
    • Suitable for: ICC/IF, ELISA, IHC-Fr
    • Reacts with: Species independent
    • Isotype: IgG

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    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-Citrulline antibody
      Citrulline 一次抗体 製品一覧
    • 製品の詳細

      Rabbit polyclonal to Citrulline
    • 由来種

      Rabbit
    • アプリケーション

      適用あり: ICC/IF, ELISA, IHC-Frmore details
    • 種交差性

      交差種: Species independent
    • 免疫原

      Chemical/ Small Molecule by a Glutaraldehyde linker.

    • 特記事項

      This polyclonal antibody recognizes only citrulline residues on proteins not free citrulline.

      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 or -80°C. Avoid repeated freeze / thaw cycles.
    • バッファー

      Constituent: Glycerol
    • Concentration information loading...
    • 精製度

      Ammonium Sulphate Precipitation
    • ポリ/モノ

      ポリクローナル
    • アイソタイプ

      IgG
    • 研究分野

      • Signal Transduction
      • Metabolism
      • Amino Acids
      • Metabolism
      • Pathways and Processes
      • Metabolic signaling pathways
      • Amino acid metabolism
      • Metabolism
      • Types of disease
      • Cancer

    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)

    アプリケーション

    Our Abpromise guarantee covers the use of ab6464 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
    ICC/IF Use at an assay dependent concentration.
    ELISA 1/5000 - 1/10000.
    IHC-Fr 1/1000 - 1/5000.

    ターゲット情報

    • 関連性

      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.

    画像

    • Immunocytochemistry/ Immunofluorescence - Anti-Citrulline antibody (ab6464)
      Immunocytochemistry/ Immunofluorescence - Anti-Citrulline antibody (ab6464)This image is courtesy of an Abreview submitted by Mohamed Bashir
      Immunofluorescence analysis of THP-1 cells, staining Citrulline with ab6464.

      Cells were fixed with paraformaldehyde and blocked with 0.05% BSA for 1 hour at 37°C. Samples were incubated with primary antibody (1/500 in diluent) for 2 hours at 37°C. ab6717 Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (FITC) was used to detect staining.

      See Abreview

    プロトコール

    • Immunohistochemistry 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 (13)

    ab6464 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。

    ab6464 は 13 報の論文で使用されています。

    • Mohamed BM  et al. Induction of protein citrullination and auto-antibodies production in murine exposed to nickel nanomaterials. Sci Rep 8:679 (2018). PubMed: 29330439
    • Verheul MK  et al. Pitfalls in the detection of citrullination and carbamylation. Autoimmun Rev 17:136-141 (2018). PubMed: 29203292
    • Walker JL  et al. In wound repair vimentin mediates the transition of mesenchymal leader cells to a myofibroblast phenotype. Mol Biol Cell 29:1555-1570 (2018). PubMed: 29718762
    • Feigenson M  et al. Histone Deacetylase 3 Deletion in Mesenchymal Progenitor Cells Hinders Long Bone Development. J Bone Miner Res 32:2453-2465 (2017). PubMed: 28782836
    • Horibata S  et al. Role of peptidylarginine deiminase 2 (PAD2) in mammary carcinoma cell migration. BMC Cancer 17:378 (2017). PubMed: 28549415
    View all Publications for this product

    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

    1-10 of 24 Abreviews or Q&A

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Citrulline antibody

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (Vein)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Citrate based (Vector Laboratories H-3300)
    Permeabilization
    Yes - 0.2% Triton-X 10 mins RT
    Specification
    Vein
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 4% · Temperature: RT°C
    Fixative
    Formaldehyde
    Read More

    Abcam user community

    Verified customer

    投稿 Jan 29 2018

    Western blot abreview for Anti-Citrulline antibody

    Average
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Recombinant protein (Histone H3)
    Gel Running Conditions
    Non-reduced Denaturing
    Loading amount
    1 µg
    Treatment
    1µg PAD4 for 24 hrs
    Specification
    Histone H3
    Blocking step
    Milk as blocking agent for 18 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
    Read More

    Mona Biermann

    Verified customer

    投稿 Sep 26 2016

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Citrulline antibody

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 37°C
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: 10mM Sodium Citrate Buffer with 0.05% Tween 20 pH 6.0
    Sample
    Human Tissue sections (Breast, DCIS tumor with apoptotic center)
    Specification
    Breast, DCIS tumor with apoptotic center
    Permeabilization
    Yes - Tris Buffered Saline with 0.025% Triton X-100
    Fixative
    Formaldehyde
    Read More

    Ms. Lynne Anguish

    Verified customer

    投稿 Jun 09 2014

    Question

    I need to stain for modified citrulline (residues) or modified citrullinated vimentin on a western blot. What is the most suitable antibody and protocol I can use? Is there an antibody I can use in both Western and IHC applications?

    Read More

    Abcam community

    Verified customer

    Asked on Sep 28 2012

    Answer

    Thank you for contacting us. We currently have two antibodies that will detect the citrulline modification on any proteins: ab6464 and ab100932. While ab6464 has been validated in IHC on frozen tissue sections, we have not tested this antibody for use in WB. Similarly, ab100932 has been validated in WB but not in IHC. If you would like to test either of these antibodies, I am happy to offer you our 100% testing discount program. If you purchase the antibody for full price and submit an Abreview with your protocol and results using the antibody in an untested application, you can get a discount code for a free primary antibody on a future purchase. Whether or not the antibody works, the next antibody you buy from us would be free. If you are interested in this program, please reply and let me know which antibody you would like to test, and I will be happy to set up the code for you.

    Read More

    Abcam Scientific Support

    Answered on Sep 28 2012

    Question

    Thanks for the reply.
    Yes I can see they do not wash before saturation. I really dont think this difference is the key to make the ab working - I can try this. I just tried both the Abs provided (ab 6464 and 100932) in westernblotting against fibrinogen and citrullinated fibrinogen - the binding pattern is not difference between these two (fibrinogen and fibrinogen containing citrulline) - only ab100932 did stain as expected. I do not think that the Abs are able to bind citrulline in all proteins as stated. I have no clue how to use the antibodies to distinguish beetween fibrinogen and fibrinogen containing citrullines. Have they only been able to distinguish between BSA and citrullinated BSA at this point?
    Best Regards

    Read More

    Abcam community

    Verified customer

    Asked on Sep 27 2012

    Answer

    Thank you for your reply and this complement of information.

    ab6464 has not been tested in Western Blot and may not be suitable for this application. This would explain why you don't see any staining with this antibody.
    The information on the product datasheet as well as the encouraging positive results obtained in Western Blot suggest that ab100932 is indeed capable of targeting the citrulline independently of the amino acid sequence.

    An optimisation of the incubation time and conditions for the antibody ab100932 in ELISA may be necessary in this case.

    Please let me know about the new trials with the antibodies in ELISA. If the problem stays the same I will be happy to issue a credit note against the original order.

    Read More

    Abcam Scientific Support

    Answered on Sep 27 2012

    Question


    You helped me out ealier regarding ab100932 [CCE4141921]. We have now got the ab6464 and it cannot distinguish between fibrinogen containing citrullines or not from our experiments - same problem.
    I have attached the protocol and the results for the indirect ELISA we did. Let me know if I should send this information otherwise.
    We not sure if this antibody can be used in this indirect setup?? We thought about coating with ab6464 - then add citrullinated fibrinogen/fibrinogen and then detect with a fibrinogen antibody - this will might work. However we are not sure if the protein does regonize citrullines since first experiment does not work, if not it, we buy a fibrinogen-antibody for no reason.
    Maybe you have suggestions?
    Best Regards

    Read More

    Abcam community

    Verified customer

    Asked on Sep 26 2012

    Answer

    Thank you for contacting us.

    The source of the antibody informed us that they have never tested the antibody by coating plates with fibrinogen.
    Their testing are made using BSA and citrunilated BSA. However, the testing conditions are very similar to yours. The difference is that the lab do not perform a washing step before the saturation with BSA.
    I attached to this email the results obtained during the validation of batch GR22466 of this antibody ab6464 as well as an ELISA protocol provided by the lab.

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

    Read More

    Abcam Scientific Support

    Answered on Sep 26 2012

    Question

    Thanks a lot for the protocol. I tried out using the ab6464 antibody and finally it gave positive results. The only modification I did was that instead of OPD I've used TMB and sulfuric acid. Thanks for the suggestion!
    Best regards,

    Read More

    Abcam community

    Verified customer

    Asked on Jul 18 2012

    Answer

    Thank you for your email.

    I am very happy to hear that the protocol optimizations improved the results and the antibody is now working fine.

    I wish all the best for future experiments and research.

    Read More

    Abcam Scientific Support

    Answered on Jul 18 2012

    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

    Abcam community

    Verified customer

    Asked on Jul 17 2012

    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.

    Read More

    Abcam Scientific Support

    Answered on Jul 17 2012

    Question

    I am also sorry about that because I have good experience with your antibodies. It was surprising for me as well that even the new antibody can not recognize the antigen ... I thought that there might be some problem in our protocol, but I can not find it. I filled out the questionery and I hope that we can figure out together what is going on ... It would be very urgent as some clinical samples are waiting for us and the deadline for the measurements is the end of June.
    1. protocol
    1) Abcam product code: ab6464
    2) Abcam order reference number or product batch number: 1092015
    3) Description of the problem: we can not get positive signal using the antibody
    4) Type of ELISA: indirect
    5) Sample details: citrullinated-peptides and citrullinated BSA
    6) Target antigen (if found in heterogeneous sample): citrullinated proteins
    7) Positive control: in house citrullinated peptides with PAD4 enzyme
    8) Negative control: PBS coating (no antigen)
    9) Blocking agent (eg BSA…):
    Concentration: 1% milk powder in TBS
    Blocking time: 1 hour
    Blocking temperature: room temperature
    10) Capture or Primary antibody information
    Used to coat wells? No
    Species: polyclonal rabbit IgG
    Reacts against: citrulline
    Concentration or dilution: 1/5000
    Diluent buffer: 0.1% milk-TTBS
    Incubation time: 1 hour
    Incubation temperature: room temperature
    11) Detection antibody:
    Species:
    Reacts against:
    Concentration or dilution:
    Diluent buffer:
    Incubation time:
    Incubation temperature:
    Conjugation:
    12) Enzyme labeled secondary antibody:
    Species: sheep polyclonal secondary IgG H&L
    Reacts against: rabbit IgG
    Concentration or dilution : 1/5000
    Diluent buffer : 0.1% milk-TTBS
    Incubation time: 1 hour
    Incubation temperature: room temperature
    Conjugation: alkaline phosphatase
    13) Washing after primary and secondary antibodies:
    Buffer: TTBS
    Number of washes: 3
    Length of washes: 10 seconds
    14) Substrate: 1 mM pNPP in 30 mM NaOH
    Incubation time: 30 minutes in dark at room temperature
    15) How many times have you run this ELISA? 6 times
    Do you obtain the same results every time? We always get negative results.
    What steps have you altered to try and optimize the use of this antibody? Different dilutions of the antibody, overnight coating, increasing the incubation time.
    2. protocol
    1) Abcam product code: ab6464
    2) Abcam order reference number or product batch number: 1092015
    3) Description of the problem: we can not get positive signal using the antibody
    4) Type of ELISA: indirect
    5) Sample details: CCP (CCPlus® Immunoscan from EuroDiagnostica)
    6) Target antigen (if found in heterogeneous sample): CCP
    7) Positive control:
    8) Negative control:
    9) Blocking agent (eg BSA…): no blocking was required
    Concentration:
    Blocking time:
    Blocking temperature:
    10) Capture or Primary antibody information
    Used to coat wells? No
    Species: polyclonal rabbit IgG
    Reacts against: citrulline
    Concentration or dilution: 1/5000
    Diluent buffer: 0.1% milk-TTBS
    Incubation time: 1 hour
    Incubation temperature: room temperature
    11) Detection antibody:
    Species:
    Reacts against:
    Concentration or dilution:
    Diluent buffer:
    Incubation time:
    Incubation temperature:
    Conjugation:
    12) Enzyme labeled secondary antibody:
    Species: sheep polyclonal secondary IgG H&L
    Reacts against: rabbit IgG
    Concentration or dilution : 1/5000
    Diluent buffer : 0.1% milk-TTBS
    Incubation time: 1 hour
    Incubation temperature: room temperature
    Conjugation: alkaline phosphatase
    13) Washing after primary and secondary antibodies:
    Buffer: TTBS
    Number of washes: 3
    Length of washes: 10 seconds
    14) Substrate: 1 mM pNPP in 30 mM NaOH
    Incubation time: 30 minutes in dark at room temperature
    15) How many times have you run this ELISA? Twice.
    Do you obtain the same results every time? Yes.
    What steps have you altered to try and optimize the use of this antibody? None
    3. protocol
    1) Abcam product code: ab6464
    2) Abcam order reference number or product batch number: 1092015
    3) Description of the problem: we can not get positive signal using the antibody
    4) Type of ELISA: indirect
    5) Sample details: CCP (CCPlus® Immunoscan from EuroDiagnostica)
    6) Target antigen (if found in heterogeneous sample): CCP
    7) Positive control:
    8) Negative control:
    9) Blocking agent (eg BSA…): no blocking was required
    Concentration:
    Blocking time:
    Blocking temperature:
    10) Capture or Primary antibody information
    Used to coat wells? No
    Species: polyclonal rabbit IgG
    Reacts against: citrulline
    Concentration or dilution: 1/5000
    Diluent buffer: Sample diluent provided with the kit
    Incubation time: 1 hour
    Incubation temperature: room temperature
    11) Detection antibody:
    Species:
    Reacts against:
    Concentration or dilution:
    Diluent buffer:
    Incubation time:
    Incubation temperature:
    Conjugation:
    12) Enzyme labeled secondary antibody:
    Species: goat polyclonal antibody
    Reacts against: rabbit IgG
    Concentration or dilution : 1/2000
    Diluent buffer : Sample diluent provided with the kit
    Incubation time: 30 minutes
    Incubation temperature: room temperature
    Conjugation: HRP
    13) Washing after primary and secondary antibodies:
    Buffer: TTBS
    Number of washes: 3
    Length of washes: 10 seconds
    14) Substrate: 100 ul TMB
    Incubation time: 30 minutes in dark at room temperature
    Stop solution: 50 ul 1 mM H2SO4
    15) How many times have you run this ELISA? Twice.
    Do you obtain the same results every time? Yes.
    What steps have you altered to try and optimize the use of this antibody? We applied 1/5000 and 1/10000 primary antibody dilutions.
    In case of CCP (protocols 2 and 3) no positive control was used. From this batch the immunological laboratory of the University routinely uses the wells for anti-citrulline antibody detection from the serum of RA patients and it works well.
    All the absorbances recorded in all protocols were less than 0.1
    Thank you for your help!
    Best regards,

    Read More

    Abcam community

    Verified customer

    Asked on Jul 03 2012

    Answer

    Thank you very much for filling in the questionnaire.

    The lab is using the belowprotocol to test the antibody. I noticed there are differences, particular concerning the buffers. The positive control is Citrulline-BSA.

    Maybe this protocol will help improve the results. Please let me know how you get on.

    ELISA protocol:

    1 - Coating of Immunogen(BSA) (10µg/ml) in maxisorp well plates with a solution of sodium carbonate buffer 0.05M (pH 9,6), during sixteen hours at 4°C.

    2 -Saturation of well plates with of a solution of PBS (pH 7,35) containing 2.5g/l of BSA , 10% and 0.5% of Tween (one hour at 37°C).

    3 - Wash with PBS containing 0.5% of Tween (PBS Tween) (three times).

    4 -Antibody will be diluted (1/2,000 – 1/10,000) in PBS Tween containing 2.5g/l BSA, 200µl by well plate (incubating during 2 hours at 37°C).

    5 - Wash with PBS Tween (three times).

    6 - 200µl of peroxidase-labelled goat anti-rabbit diluted (1/10,000) in a solution of PBS Tween containing 1g/l of BSA, will be applied by well plate (during one hour at 37°C)

    7 - Well plates will be rinsed with PBS Tween (three times).

    8 - And finally the peroxidase will be developed by incubating 200µl by well plate of a citrate 0,1M/phosphate 0,2M (pH 5) solution containing 0.4% of OPD and 0.03% of hydrogen peroxide for ten minutes in the dark, after that, we will stop the reaction by the addition of 50µl of 2M HCl.

    9 - The optical density will be measured at 492nm.

    Competitive ELISA protocol:

    The same protocol is used, but modifications occur in stage “4”. The day before the test, competition scales are made with each competitor (10-1 - 10-10g/l of BSA conjuguate) and diluted antibody. These tubes are incubated at 4°C overnight. The results are expressed as OD with competitor/ OD without competitor for each point of competition scale.

    Read More

    Abcam Scientific Support

    Answered on Jul 03 2012

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Citrulline antibody

    Poor
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (Skin)
    Specification
    Skin
    Fixative
    Formaldehyde
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: EDTA pH 9.0
    Permeabilization
    No
    Blocking step
    Casein as blocking agent for 15 minute(s) · Concentration: 0.25% · Temperature: RT°C
    Read More

    Abcam user community

    Verified customer

    投稿 Jun 15 2012

    1-10 of 24 Abreviews or Q&A

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