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Immunology Innate Immunity Macrophage / Inflamm.
使用敲除细胞株进行验证重组RabMAb

重组Anti-Lysozyme抗体[EPR2994(2)] (ab108508)

  • Datasheet
  • SDS
Reviews (3)Q&A (4)References (50)

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Western blot - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
  • Western blot - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
  • Immunocytochemistry/ Immunofluorescence - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
  • Western blot - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
  • Western blot - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
  • Western blot - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
  • Anti-Lysozyme antibody [EPR2994(2)] (ab108508)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR2994(2)] to Lysozyme
  • Suitable for: ICC/IF, WB, IHC-P
  • Knockout validated
  • Reacts with: Mouse, Human

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概述

  • 产品名称

    Anti-Lysozyme抗体[EPR2994(2)]
    参阅全部 Lysozyme 一抗
  • 描述

    兔单克隆抗体[EPR2994(2)] to Lysozyme
  • 宿主

    Rabbit
  • Tested Applications & Species

    Application Species
    ICC/IF
    Human
    IHC-P
    Mouse
    Human
    WB
    Mouse
    Human
    See all applications and species data
  • 免疫原

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • 阳性对照

    • WB: THP-1, HepG2, RAW 264.7 and HL-60 whole cell lysate; Human spleen tissue lysate; Natural human Lysozyme protein. IHC-P: Human tonsil, spleen, lung, kidney, brain, breast and heart tissues; Mouse spleen and small intestine tissues. ICC/IF: THP-1 cells.
  • 常规说明

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

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

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.

    One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.

    Learn more here.

性能

  • 形式

    Liquid
  • 存放说明

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • 存储溶液

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 40% Glycerol, 0.05% BSA, PBS
  • Concentration information loading...
  • 纯度

    Protein A purified
  • 克隆

    单克隆
  • 克隆编号

    EPR2994(2)
  • 同种型

    IgG
  • 研究领域

    • Immunology
    • Innate Immunity
    • Macrophage / Inflamm.
    • Immunology
    • Adaptive Immunity
    • T Cells
    • Cytotoxic Cells
    • Cardiovascular
    • Blood
    • Other
    • Tags & Cell Markers
    • Subcellular Markers
    • Organelles
    • Lysosome

相关产品

  • Alternative Versions

    • Anti-Lysozyme antibody [EPR2994(2)] - BSA and Azide free (ab185129)
    • Biotin Anti-Lysozyme antibody [EPR2994(2)] (ab197704)
    • HRP Anti-Lysozyme antibody [EPR2994(2)] (ab197705)
  • Compatible Secondaries

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

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Positive Controls

    • HL-60 whole cell lysate (ab7914)
  • Recombinant Protein

    • Recombinant Human Lysozyme protein (ab158839)
  • Related Products

    • Prestained Protein Ladder - Broad molecular weight (10-245 kDa) (ab116028)

应用

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab108508 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

应用 Species
ICC/IF
Human
IHC-P
Mouse
Human
WB
Mouse
Human
应用 Ab评论 说明
ICC/IF
1/250.

 For unpurified use at 1/500 - 1/1000.

WB
1/10000 - 1/50000. Predicted molecular weight: 17 kDa.
IHC-P (3)
1/1000 - 1/5000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Heat up to 98°C, below boiling, and then let cool for 10-20 min.

See IHC antigen retrieval protocols.

说明
ICC/IF
1/250.

 For unpurified use at 1/500 - 1/1000.

WB
1/10000 - 1/50000. Predicted molecular weight: 17 kDa.
IHC-P
1/1000 - 1/5000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Heat up to 98°C, below boiling, and then let cool for 10-20 min.

See IHC antigen retrieval protocols.

  • 应用说明
    Is unsuitable for IP.
  • 靶标

    • 功能

      Lysozymes have primarily a bacteriolytic function; those in tissues and body fluids are associated with the monocyte-macrophage system and enhance the activity of immunoagents.
    • 疾病相关

      Amyloidosis 8
    • 序列相似性

      Belongs to the glycosyl hydrolase 22 family.
    • 细胞定位

      Secreted.
    • Target information above from: UniProt accession P61626 The UniProt Consortium
      The Universal Protein Resource (UniProt) in 2010
      Nucleic Acids Res. 38:D142-D148 (2010) .

      Information by UniProt
    • 数据库链接

      • Entrez Gene: 4069 Human
      • Entrez Gene: 17105 Mouse
      • Entrez Gene: 17110 Mouse
      • Omim: 153450 Human
      • SwissProt: P61626 Human
      • SwissProt: P08905 Mouse
      • Unigene: 524579 Human
      • 别名

        • 1 4 beta N acetylmuramidase C antibody
        • 1 antibody
        • 4-beta-N-acetylmuramidase C antibody
        • EC 3.2.1.17 antibody
        • LYSC_HUMAN antibody
        • Lysosyme antibody
        • Lysozyme (renal amyloidosis) antibody
        • Lysozyme C antibody
        • Lysozyme C precursor antibody
        • LYZ antibody
        • LZM antibody
        • Renal amyloidosis antibody
        see all

      图片

      • Western blot - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Western blot - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        All lanes : Anti-Lysozyme antibody [EPR2994(2)] (ab108508) at 1/10000 dilution

        Lane 1 : Wild-type THP1 cell lysate
        Lane 2 : LYZ knockout THP1 cell lysate
        Lane 3 : HepG2 cell lysate
        Lane 4 : HeLa cell lysate

        Lysates/proteins at 20 µg per lane.

        Performed under reducing conditions.

        Predicted band size: 17 kDa
        Observed band size: 16 kDa why is the actual band size different from the predicted?



        Lanes 1 - 4: Merged signal (red and green). Green - ab108508 observed at 16 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

        ab108508 was shown to react with Lysozyme in wild-type THP1 cells in Western blot with loss of signal observed in LYZ knockout sample.Wild-type THP1 and LYZ knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab108508 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)

        Immunohistochemical analysis of paraffin-embedded human lung tissue sections labeling lysozyme with purified ab108508 at a dilution of 1/1500 (0.6 μg/ml). ab97051 Goat Anti-Rabbit IgG H&L (HRP) at 1/500 was used as the secondary anitbody.Sections were counterstained with Hematoxylin. Antigen retrieval was heat mediated using EDTA Buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

      • Western blot - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Western blot - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Anti-Lysozyme antibody [EPR2994(2)] (ab108508) at 1/10000 dilution (purified) + RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 20 µg

        Secondary
        Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

        Predicted band size: 17 kDa
        Observed band size: 16 kDa why is the actual band size different from the predicted?



        Blocking/Diluting buffer 5% NFDM/TBST
      • Immunocytochemistry/ Immunofluorescence - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Immunocytochemistry/ Immunofluorescence - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)

        Immunocytochemistry/Immunofluorescence analysis of THP-1 (Human monocytic leukemia) cells labeling lysozyme with purified ab108508 at 1/250. Cells were fixed with 100% methanol. ab150077, Alexa Fluor®488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Cells were counter-stained with ab195889 Anti-Alpha Tubulin antibody [DM1A] (1/200, 2.5 g/mL) - Microtubule Marker (Alexa Fluor®594) at 1/200. DAPI (blue) was used as a nuclear counterstain. Secondary Only Control: PBS was used instead of the primary antibody as the negative control.

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)This image is courtesy of an Abreview submitted by Mr. Thorsten Cramer.

        Paraffin-embedded mouse small intestine tissue stained for Lysozyme using ab108508 in immunohistochemical analysis.

        See Abreview

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)

        Immunohistochemical analysis of paraffin-embedded mouse spleen tissue sections labeling lysozyme with purified ab108508 at a dilution of 1/1500 (0.6 μg/ml). ab97051 Goat Anti-Rabbit IgG H&L (HRP) at 1/500 was used as the secondary anitbody. Sections were counterstained with hematoxylin. Antigen retrieval was heat mediated using EDTA Buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

      • Western blot - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Western blot - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        All lanes : Anti-Lysozyme antibody [EPR2994(2)] (ab108508) at 1/10000 dilution (unpurified)

        Lane 1 : HL60 (human promyelocytic leukemia cell line) cell lysate
        Lane 2 : Human spleen lysate

        Lysates/proteins at 10 µg per lane.

        Predicted band size: 17 kDa

      • Western blot - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Western blot - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Anti-Lysozyme antibody [EPR2994(2)] (ab108508) at 1/50000 dilution (purified) + HL-60 (Human promyelocytic leukemia cell line) whole cell lysate at 20 µg

        Secondary
        Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

        Predicted band size: 17 kDa
        Observed band size: 16 kDa why is the actual band size different from the predicted?



        Blocking/Diluting buffer 5% NFDM/TBST
      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)

        Unpurified ab108508, at 1/1000 dilution, staining Lysozyme in Human tonsil by Immunohistochemistry, Paraffin-embedded tissue.

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

      • Western blot - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Western blot - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Anti-Lysozyme antibody [EPR2994(2)] (ab108508) at 1 µg/ml (unpurified) + Native human Lysozyme protein (ab91125) at 0.1 µg

        Secondary
        Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

        Developed using the ECL technique.

        Performed under reducing conditions.

        Predicted band size: 17 kDa


        Exposure time: 4 minutes
      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)

        Unpurified ab108508, at 1/1000 dilution, staining Lysozyme in Human spleen by Immunohistochemistry, Paraffin-embedded tissue.

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

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)

        Unpurified ab108508, at 1/1000 dilution, staining Lysozyme in Human kidney by Immunohistochemistry, Paraffin-embedded tissue.

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

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)

        Unpurified ab108508 showing negative staining in Normal human brain tissue.

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

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)

        Unpurified ab108508 showing negative staining in Normal human breast tissue.

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

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lysozyme antibody [EPR2994(2)] (ab108508)

        Unpurified ab108508 showing negative staining in Normal human heart tissue.

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

      • Anti-Lysozyme antibody [EPR2994(2)] (ab108508)
        Anti-Lysozyme antibody [EPR2994(2)] (ab108508)

      实验方案

      • Western blot protocols
      • Immunohistochemistry protocols
      • Immunocytochemistry & immunofluorescence protocols

      Click here to view the general protocols

      数据表及文件

      • SDS download

      • Datasheet download

        Download

      文献 (50)

      发表研究结果有使用 ab108508?请让我们知道,以便我们可以引用本数据表中的参考文章。

      ab108508 被引用在 50 文献中.

      • Xie Y  et al. Impact of a high-fat diet on intestinal stem cells and epithelial barrier function in middle-aged female mice. Mol Med Rep 21:1133-1144 (2020). PubMed: 32016468
      • Bergkvist L  et al. Serum amyloid P component promotes formation of distinct aggregated lysozyme morphologies and reduces toxicity in Drosophila flies expressing F57I lysozyme. PLoS One 15:e0227227 (2020). PubMed: 31978114
      • He D  et al. Gut stem cell aging is driven by mTORC1 via a p38 MAPK-p53 pathway. Nat Commun 11:37 (2020). PubMed: 31896747
      • Wang Y  et al. Single-cell transcriptome analysis reveals differential nutrient absorption functions in human intestine. J Exp Med 217:N/A (2020). PubMed: 31753849
      • Pérez-Debén S  et al. iTRAQ comparison of proteomic profiles of endometrial receptivity. J Proteomics 203:103381 (2019). PubMed: 31102758
      View all Publications for this product

      客户评价及客户问答

      Show All 评价 Q&A
      提交评价 提交问题

      1-7 of 7 Abreviews or Q&A

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Lysozyme antibody [EPR2994(2)]

      Excellent
      Abreviews
      Abreviews
      abreview image
      Application
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
      Sample
      Mouse Tissue sections (Small intestine)
      Antigen retrieval step
      Heat mediated - Buffer/Enzyme Used: Citrate (pH 6.0)
      Permeabilization
      No
      Specification
      Small intestine
      Blocking step
      Serum as blocking agent for 30 minute(s) · Concentration: 2.5% · Temperature: 25°C
      Fixative
      Formaldehyde
      Read More

      Mr. Brett Bell

      Verified customer

      提交于 Dec 28 2020

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Lysozyme antibody [EPR2994(2)]

      Excellent
      Abreviews
      Abreviews
      abreview image
      Application
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
      Sample
      Rat Tissue sections (Spleen)
      Antigen retrieval step
      Heat mediated - Buffer/Enzyme Used: Citrate pH 6.0
      Permeabilization
      No
      Specification
      Spleen
      Blocking step
      Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 21°C
      Fixative
      Paraformaldehyde
      Read More

      Abcam user community

      Verified customer

      提交于 Jul 31 2018

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Lysozyme antibody [EPR2994(2)]

      Excellent
      Abreviews
      Abreviews
      abreview image
      Application
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
      Sample
      Mouse Tissue sections (small intestine)
      Antigen retrieval step
      Heat mediated - Buffer/Enzyme Used: citrate
      Permeabilization
      No
      Specification
      small intestine
      Blocking step
      AB block Dako X0590 as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: RT°C
      Fixative
      10% NBF
      Read More

      Mr. Thorsten Cramer

      Verified customer

      提交于 Aug 18 2016

      Question

      The product data sheet says it reacts with both mouse and human, but what I want to know is if it discriminiates between the mouse M or P lysozyme isoform?

      Read More

      Abcam community

      Verified customer

      Asked on Feb 08 2013

      Answer

      This antibody would not distinguish between the mouse M or P lysozyme isoforms as the immunogen sequence in conserved in both.

      Read More

      Abcam Scientific Support

      回复于 Feb 08 2013

      Question

      I contacted you earlier today for a request
      about antibodies reactive against different lysozyme forms.
      Basically I am working with human lysozyme and its two mutants.the sequence
      of WT lysozyme is given here
      http://www.uniprot.org/uniprot/P61626
      the mutants are
      1) I 74 substituted to T
      2) T 88 substituted to N
      please let me know if any of the monoclonal antibodies to lysozyme: e.g.
      ab91653 or ab108508 are expected to be reactive to all three forms. If you
      think there is such an antibody available please let me know what it was
      raised against.

      Read More

      Abcam community

      Verified customer

      Asked on Aug 29 2012

      Answer

      Thank you for providing that information.

      I have had a look at the two rabbit monoclonal against Lysozyme (ab91653 and ab108508). Both of these antibodies should be able to detect all three forms of the protein (the wild type as well as your two mutants). The immunogen used to raise the ab108508 antibody has been taken from a region N-terminal of your mutations, whilst the immunogen used to raise the antibody ab91653 is from the C-terminal domain. Neither immunogen overlaps any of the mutation sites.

      I hope this information has beenof help. If you have any further questions please do not hesitate to ask.

      Read More

      Abcam Scientific Support

      回复于 Aug 29 2012

      Question

      Please can you send me the IHC protocol for this antibody.

      Read More

      Abcam community

      Verified customer

      Asked on May 30 2012

      Answer


      Thank you for contacting us yesterday.

      I have received further information from the lab regarding the IHC protocol for ab108508. I can
      confirm that 0.01M Sodium Citrate Buffer, pH 6.0 was used for antigen retrieval. Here is a copy
      of the protocol:

      Immunohistochemistry Protocol for Paraffin-embedded Tissues

      1. Solutions and reagents
      1.1. Xylene
      1.2. Ethanol, anhydrous denatured, histological grade (100%, 95%, 70%, 50%)
      1.3. Washing buffer/TBST: 1X TBS/0.1% Tween-20, pH to 7.6.
      1.4. Distilled water (dH2O)
      1.5. Antigen Retrieval Solution: 0.01M Sodium Citrate Buffer, pH 6.0
      To prepare Antigen Retrieval stock solutions:
      10X Stock: Dissolve 29.4 g sodium citrate trisodium salt dehydrate (C 6H 5Na 3O 7 2H2O in 1 liter of dH2O. Add 5mL Tween-20.
      1X Working Solution: Mix 200mL 10X stock with 1800mL dH2O; pH to 6.0
      1.6. 3% Hydrogen Peroxide
      1.7. Blocking Buffer: 10% serum in PBS (serum origin depends on the host of the secondary antibody)
      1.8. Primary Antibody Diluent: 5% serum in PBS (serum origin depends on the host of the
      Secondary antibody)
      1.9. Hematoxylin
      1.10. Permanent Mounting Medium

      2. Protocol
      2.1. Deparaffinization/Rehydration
      2.1.1. Heat slides in an oven at 65 °C for 1 hour.
      2.1.2. De-paraffinize/hydrate using the following series of washes: two Xylene washes (3 min each), followed by two 100% ethanol rinses (3 min each), followed by 95% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, followed by TBST wash for 3 min on a shaker.

      2.2. Antigen Retrieval
      This is recommended Heat Induced Epitope Retrieval (HIER) using Decloaking
      Chamber/Pressure Cooker. Hot water bath or Microwave with temperature sensor can be also used
      (protocol would vary depending on the method used).
      2.2.1. Add 500 ml of dH 2O to Decloaker/Pressure Cooker.
      2.2.2. Immerse slides into staining dish containing Antigen Retrieval Solution. Place staining dish into decloaking chamber.
      2.2.3. Program to run for 30 seconds at 125° C, followed by 10 seconds at 90° C.
      2.2.4. Let it cool down to room temperature (10 – 20 minutes).
      2.2.5. Removes slides and rinse in TBST.
      2.2.6. Proceed to Staining step.

      2.3. Staining
      2.3.1. Wash slides with TBST for 3 min on a shaker.
      2.3.2. Inactivate endogenous peroxidase by covering tissue with 3% hydrogen peroxide for 5 min.
      2.3.3. Wash slides three times with TBST (3 min each on a shaker).
      2.3.4. Block slides with the blocking solution for 1 hour.
      2.3.5. Dilute primary antibody in primary antibody diluent per recommendation on data sheet.
      2.3.6. Apply primary antibody to each section and incubate overnight in the humidified chamber (4 ºC).
      2.3.7. Wash slides three times with TBST (3 min each on a shaker).
      2.3.8. Apply to each section secondary HRP-conjugated anti-rabbit antibody diluted in the blocking solution per manufacturer’s recommendation; incubate for 30 min at room temperature.
      2.3.9. Wash slides three times with TBST (5 min each on a shaker).
      2.3.10. Add freshly prepared DAB substrate to the sections and incubate until stain develops (generally 1 min).
      2.3.11. Rinse sections with water.
      2.3.12. Counterstain with Hematoxylin (generally 10 seconds).
      2.3.13. Rinse sections with water.
      2.3.14. Dehydrate samples using two washes with 100% Ethanol (3 min each), followed by
      two rinses with Xylene (3 min each).
      2.3.15. Mount coverslips on slides using permanent mounting medium.

      I hope this information helps and that the antibody works well. Please do not hesitate to contact us should you have further problems.

      Read More

      Abcam Scientific Support

      回复于 May 30 2012

      Question

      For anti-lysozyme antibody ab91563 and ab108508, Which one will react with human but not cross-react with chicken?

      Read More

      Abcam community

      Verified customer

      Asked on Dec 28 2011

      Answer

      Thank you for contacting us. Based on sequence analysis, immunogen of ab91653 does not share any significant sequence similarity with chicken. For the immunogen of ab108508, it shares 67% identity with chicken Lysozyme C, so it should also be human specific. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

      Read More

      Abcam Scientific Support

      回复于 Dec 28 2011

      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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