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Epigenetics and Nuclear Signaling Histones Variants
使用敲除细胞株进行验证

Anti-Histone H1.2抗体- ChIP Grade (ab4086)

  • Datasheet
  • SDS
Reviews (2)Q&A (6)References (19)

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Western blot - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)
  • Western blot - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)
  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)
  • Western blot - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)
  • Immunoprecipitation - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)
  • ChIP - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)

Key features and details

  • Rabbit polyclonal to Histone H1.2 - ChIP Grade
  • Suitable for: ChIP, WB, IP, ICC/IF, IHC-P
  • Knockout validated
  • Reacts with: Human
  • Isotype: IgG

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概述

  • 产品名称

    Anti-Histone H1.2抗体- ChIP Grade
    参阅全部 Histone H1.2 一抗
  • 描述

    兔多克隆抗体to Histone H1.2 - ChIP Grade
  • 宿主

    Rabbit
  • 特异性

    This antibody has only been tested on bulk HeLa histones. We expect this antibody to be specific for Histone H1.2 - however we have not tested this specifically on different histone H1 variants.
  • 经测试应用

    适用于: ChIP, WB, IP, ICC/IF, IHC-Pmore details
  • 种属反应性

    与反应: Human
  • 免疫原

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
    (Peptide available as ab16936)

  • 阳性对照

    • HeLa Histone preparation
  • 常规说明

    Histone H1.2 is one of five known h1 variants (known as H1.1/2/3/4/5 and/or H1.a/b/c/d/e). The H1 variants differ from each other in the amino acid sequence of their N-terminal regions.

    For Western Blotting, Abcam recommends blocking in milk in order to increase band clarity.

    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 this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. 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, along with publications, customer reviews and Q&As

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 or -80°C. Avoid freeze / thaw cycle.
  • 存储溶液

    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituent: PBS

    Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
  • Concentration information loading...
  • 纯度

    Immunogen affinity purified
  • Primary antibody说明

    Histone H1.2 is one of five known h1 variants (known as H1.1/2/3/4/5 and/or H1.a/b/c/d/e). The H1 variants differ from each other in the amino acid sequence of their N-terminal regions.
  • 克隆

    多克隆
  • 同种型

    IgG
  • 研究领域

    • Epigenetics and Nuclear Signaling
    • Histones
    • Variants
    • Epigenetics and Nuclear Signaling
    • DNA / RNA
    • DNA Damage & Repair
    • DNA Damage Response
    • DNA Damage Recognition

相关产品

  • ChIP Related Products

    • Rabbit Anti-Mouse IgG H&L (ab46540)
    • ChIP Kit (ab500)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
  • Isotype control

    • Rabbit IgG, polyclonal - Isotype Control (ChIP Grade) (ab171870)
  • KO cell lines

    • Human HIST1H1C (Histone H1.2) knockout A549 cell line (ab261873)
  • KO cell lysates

    • Human HIST1H1C (Histone H1.2) knockout A549 cell lysate (ab261682)
  • Recombinant Protein

    • Recombinant Human Histone H1.2 protein (ab158628)
  • Related Products

    • Anti-Histone H1.2 antibody (ab17677)

应用

The Abpromise guarantee

Abpromise™承诺保证使用ab4086于以下的经测试应用

“应用说明”部分 下显示的仅为推荐的起始稀释度;实际最佳的稀释度/浓度应由使用者检定。

应用 Ab评论 说明
ChIP
Use 2-6 µg for 25 µg of chromatin.
WB (2)
1/500. Detects a band of approximately 25 kDa (predicted molecular weight: 21 kDa).

Blocking in milk is recommended.

IP
Use a concentration of 5 µg/ml.
ICC/IF
Use a concentration of 1 µg/ml.
IHC-P
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
说明
ChIP
Use 2-6 µg for 25 µg of chromatin.
WB
1/500. Detects a band of approximately 25 kDa (predicted molecular weight: 21 kDa).

Blocking in milk is recommended.

IP
Use a concentration of 5 µg/ml.
ICC/IF
Use a concentration of 1 µg/ml.
IHC-P
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

靶标

  • 功能

    Histones H1 are necessary for the condensation of nucleosome chains into higher order structures.
  • 序列相似性

    Belongs to the histone H1/H5 family.
    Contains 1 H15 (linker histone H1/H5 globular) domain.
  • 细胞定位

    Nucleus. Chromosome.
  • Target information above from: UniProt accession P16403 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: 3006 Human
    • Omim: 142710 Human
    • SwissProt: P16403 Human
    • Unigene: 7644 Human
    • 别名

      • H1 histone family member 2 antibody
      • H1.a antibody
      • H12_HUMAN antibody
      • H1F2 antibody
      • H1s-1 antibody
      • HIST1H1C antibody
      • Histone 1 H1c antibody
      • Histone cluster 1 H1c antibody
      • Histone H1.2 antibody
      • Histone H1c antibody
      • Histone H1d antibody
      • Histone H1s-1 antibody
      • MGC3992 antibody
      see all

    图片

    • Western blot - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)
      Western blot - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)
      All lanes : Anti-Histone H1.2 antibody - ChIP Grade (ab4086) at 1/500 dilution

      Lane 1 : Wild-type HeLa cell lysate
      Lane 2 : HIST1H1C knockout HeLa cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Predicted band size: 21 kDa
      Observed band size: 37 kDa why is the actual band size different from the predicted?



      False colour image of Western blot: Anti-Histone H1.2 antibody - ChIP Grade staining at 1/500 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab4086 was shown to bind specifically to Histone H1.2. A band was observed at 37 kDa in wild-type HeLa cell lysates with no signal observed at this size in HIST1H1C knockout cell line ab261794 (knockout cell lysate ab257218). To generate this image, wild-type and HIST1H1C knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

    • Western blot - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)
      Western blot - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)
      All lanes : Anti-Histone H1.2 antibody - ChIP Grade (ab4086) at 1/500 dilution

      Lane 1 : Wild-type A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg/ml
      Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg/ml
      Lane 3 : HIST1H1C knockout A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

      Performed under reducing conditions.

      Predicted band size: 21 kDa



      Lanes 1 - 3: Merged signal (red and green). Green - ab4086 observed at 30 kDa. Red - loading control, ab130007, observed at 130 kDa.

      ab4086 was shown to recognize HIST1H1C in wild-type A549 cells as signal was lost at the expected MW in HeLa knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and HeLa knockout samples were subjected to SDS-PAGE. Ab4086 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/500 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-Histone H1.2 antibody - ChIP Grade (ab4086)
      Immunocytochemistry/ Immunofluorescence - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)

      ICC/IF image of ab4086 stained human HeLa cells. The cells were PFA fixed (3.7% PFA, 10 min) and incubated with the antibody (ab4086, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

    • Western blot - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)
      Western blot - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)

      ab4086 Histone H1.2

      The antibody was used at a dilution of 1/500

      Lane 1: HeLa Histone (5ug) + ab4086 
      Lane 2: HeLa Histone (5ug) + ab4086 + 1µg/ml of peptide (Histone H1.2) (ab16936)

      Secondary ab: Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed ab7090 (1/5000)

      Exposure time: 1 minute
      Expected molecular weight: 21.3 kDa

      ab4086 Histone H1.2

      The antibody was used at a dilution of 1/500

      Lane 1: HeLa Histone (5ug) + ab4086
      Lane 2: HeLa Histone (5ug) + ab4086 + 1µg/ml of peptide (Histone H1.2) (ab16936)

      Secondary ab: Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed ab7090 (1/5000)

      Exposure time: 1 minute
      Expected molecular weight: 21.3 kDa
    • Immunoprecipitation - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)
      Immunoprecipitation - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)

      Histone H1.2 - ChIP Grade was immunoprecipitated using 0.5mg HeLa whole cell extract, 5µg of Rabbit polyclonal to and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

      The antibody was incubated under agitation with Protein G beads for 10min, HeLa whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

      Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab4086.

      Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

      Band: 21kDa; Histone H1.2 - ChIP Grade

    • ChIP - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)
      ChIP - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)

      Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 2µg of  ab4086 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.
          

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)
      IHC image of Histone H1.2 staining in human breast carcinoma FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab4086, 1µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    实验方案

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    数据表及文件

    • SDS download

    • Datasheet download

      Download

    文献 (19)

    发表研究结果有使用 ab4086?请让我们知道,以便我们可以引用本数据表中的参考文章。

    ab4086 被引用在 19 文献中.

    • Ferrari R  et al. TFIIIC Binding to Alu Elements Controls Gene Expression via Chromatin Looping and Histone Acetylation. Mol Cell 77:475-487.e11 (2020). PubMed: 31759822
    • Teif VB  et al. Linker histone epitopes are hidden by in situ higher-order chromatin structure. Epigenetics Chromatin 13:26 (2020). PubMed: 32505195
    • Shimada M  et al. Gene-Specific H1 Eviction through a Transcriptional Activator?p300?NAP1?H1 Pathway. Mol Cell 74:268-283.e5 (2019). PubMed: 30902546
    • Scicchitano S  et al. The stem cell-associated transcription co-factor, ZNF521, interacts with GLI1 and GLI2 and enhances the activity of the Sonic hedgehog pathway. Cell Death Dis 10:715 (2019). PubMed: 31558698
    • Chiarella E  et al. ZNF521 Has an Inhibitory Effect on the Adipogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells. Stem Cell Rev N/A:N/A (2018). PubMed: 29938352
    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

    1-8 of 8 Abreviews or Q&A

    Western blot abreview for Anti-Histone H1.2 antibody - ChIP Grade

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Spermophilus tridecemlineatus Tissue lysate - whole (Liver)
    Gel Running Conditions
    Reduced Denaturing (12.5%)
    Loading amount
    15 µg
    Specification
    Liver
    Blocking step
    BSA as blocking agent for 7 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 4°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    MR. James Bjork

    Verified customer

    提交于 Aug 24 2015

    Western blot abreview for Anti-Histone H1.2 antibody - ChIP Grade

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (MRC5VA)
    Loading amount
    15 µg
    Specification
    MRC5VA
    Gel Running Conditions
    Reduced Denaturing (12%)
    Blocking step
    Milk as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    提交于 Sep 01 2009

    Question

    ab4068: lot GR74608
    4 human breast cancer cell line lysates
    sees band at expected MW, but also sees band at 15 kDa which is very strong and other bands too
    needs to use Ab in ChIP and is concerned that other protein(s) will be pulled down as well
    Ab: 1/500 o/n
    block: 5% milk
    2nd: AP + ECF (fluorescence signal), works well, priamry crtl: no signal detected

    ab5212: lot 86354
    4 human breast cancer cell line lysates
    sees band at expected MW, but also sees band at 15 kDa which is very strong and other bands too
    needs to use Ab in ChIP and is concerned that other protein(s) will be pulled down as well
    Ab: 1/500 o/n
    block: 5% milk
    2nd: AP + ECF (fluorescence signal), works well, priamry crtl: no signal detected

    Read More

    Abcam community

    Verified customer

    Asked on May 25 2012

    Answer

    Thank you for contacting us.

    Your credit note ID is xxx.

    I am sorry that these 2 antibodies did not perform as stated on the datasheet. I have asked our accounting department to issue a credit note for you, which can be redeemed against the invoice of a future order by passing it on to your purchasing department. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used. If you have questions on how to use the credit note, please contact our accounting department.

    Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department.

    The credit note ID is for your reference only and does not automatically guarantee the credit.

    I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

    Read More

    Abcam Scientific Support

    回复于 May 25 2012

    Question

    human breast cancer cells
    20 ug to 140 ug protein loaded
    transfer OK (other protein detected)
    Ab: 1/1000, 1/500 o/n 4 degree
    block: 5% and 2% milk + TBST
    2nd: works well
    tried both vials

    ab5212: no signal, but weak band at 80 kDa
    ab4086: no band at all

    Read More

    Abcam community

    Verified customer

    Asked on May 21 2012

    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 xxx for1 vial of ab4086 with a new lot. As for ab5212, order xxx has been arranged with 1x vial oflot 863574. Theestimated delivery date for this vial is May 25th. I apologize about this delay.

    Please let me know how each lot is working out for you and I will send you the second vial for each antibody.

    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, and look forward to hear back from you.

    Read More

    Abcam Scientific Support

    回复于 May 21 2012

    Question

    We require an histone H1.2 antibody, for mouse and IHC-Fr.

    Read More

    Abcam community

    Verified customer

    Asked on May 17 2012

    Answer

    Thank you very much for your telephone enquiry.

    I am sorry to confirm that to our knowledge, the two histone H1.2 antibodies we have are regrettably not tested and guaranteed in mouse or frozen sections (IHC-Fr).

    ab17677 Anti-Histone H1.2 antibody
    Rabbit polyclonal
    ICC/IF, IHC-P, IP, WB
    Reacts with:Human

    ab4086 Anti-Histone H1.2 antibody - ChIP Grade
    Rabbit polyclonal
    ChIP, ICC/IF, IHC-P, WB
    Reacts with: Human

    I have checked the alignment of the immunogen for these antibodies with the mouse Histone H1.2 protein. This is very quite low at 63%. Unfortunately, this means I would suggest there is likely to be little crossreactivity of these antibodies with the mouse histone H1.2 protein.

    Therefore, unfortunately it is not possible for use to offer any testing discount on this occasion.

    If I am sorry we have no suitably tested Histone H1.2 antibodies for your requirements on this occasion. If you have any further questions, please do not hesitate to contact us.

    Read More

    Abcam Scientific Support

    回复于 May 17 2012

    Question

    Respected sir/madam, i attended your seminar held on 16-11-11 in JNU(India) on "optimisation techniques for immunohistochemistry and introduction to ChIP".  I had some questions which were left unanswered by the speaker in that conference. I was asked to send my inquiry to you so that it could be forwarded to an expert , possibly they will be able to sort out this.   Actually I am trying to perform ChIP on patient tissue samples. Till now only limited no. of literature is available that has performed ChIP on any tissue sample. i compared all procedures/protocols available on net and tried the best among these to start my ChIP experiment, i further modified my steps according to the problem encountered and was able to standarise it till sonication step. but the problem is that my sonication condition that yielded me the product of my interest is very high . I used UP100H sonicator and the sonication condition finally used are. 100% Amplitude, 20 SEC ON,1Min OFF (total 25 cycles). but i am expecting that this high sonication condition and for such a long time may generate heat that may destroy  my protein of interest , so is it good enough to proceed to the next step as the ChIP grade antibodies recognize native strucure so how to move furher,,,,,,   any suggestions are welcomed....      

    Read More

    Abcam community

    Verified customer

    Asked on Nov 18 2011

    Answer

    Thank you for contacting us. We are pleased to hear that you attended our seminar on the 16th and hope you enjoyed it. I appreciate the time you have spent in the laboratory and understand your concerns. We would like to investigate this particular case further for you and offer some suggestions to help optimise the results for your patient tissue samples. Have you tried to keep the samples on ice during the sonication procedure? For such a long sonication it is recommended to cool the samples throughout the cycles, repeatedly adding fresh ice if necessary. Also, do you cross-link your samples? The crosslinking in ChIP serves to create a snapshot of the cell at the moment when you add the formaldehyde, as it kills cells immediately and crosslinks protein to DNA. This step also has influence on the sonication efficiency. For details please find our protocol attached. May I ask if this sonication step has been optimised? Ideally, DNA fragments should be approximately 300 bp in length and it is advisable to perform a time course to determine the optimal sonication conditions. So depending on the sample type a shorter procedure may be suitable as well. Although some ChIP grade antibodies may recognize the native structure of the antigen, I would not recommend proceeding to the next step without cross-linking and sonicating the samples. I hope this information is helpful and would like to wish you good luck with your research. The technical team is always at your service, should you require further expert advice. PS: I am happy to let you know that we have a webinar on ChIP coming up (presumably in January) that might be of interest to you. Details can be found in due course on our website (https://www.abcam.com/index.html?pageconfig=tradeshow).

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    Abcam Scientific Support

    回复于 Nov 18 2011

    Question

    Was this antibody tested in Western blot on purified HeLa histones, enriched HeLa histones, or HeLa extract? If it was tested on enriched histones, could you tell me the protocol?

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    Abcam community

    Verified customer

    Asked on Feb 22 2006

    Answer

    Thank you for your enquiry. This antibody was tested on purified core histones purchased from another company. They were purified by acid extraction precipitation from log phase untreated HeLa cells. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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    Abcam Scientific Support

    回复于 Feb 23 2006

    Question

    I'm working with your antibody anti Histone H1.2 (ab 4086)using WB technique with nice results. I obtain with my samples a band of aproximately 25 kDa as it is shown in your web. I would like to test the specificity of this band with your peptide Histone H1.2 (ab 16936)and I would like to know which is the protocol you could recommend me as in the web there are few details. Thank you.

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    Abcam community

    Verified customer

    Asked on Apr 25 2005

    Answer

    Thank you for your e-mail. We are very pleased to hear that you are satisfied with the antibody ab4086. I enclose our recommended protocol for blocking, please do not hesitate to contact us again if you need further information. In principle, a small volume of antibody (e.g., 1-5 ul) is first reacted with excess peptide (5-50 fold over the antibody; e.g., 1 ug antibody reacted with 5-50 ug peptide; exact amounts determined by titration) to neutralize it. The neutralized antibody can no longer subsequently bind to another antigen (a band of interest) or staining pattern. So the band(s)/staining that is competed by the antigen/peptide is supposedly specific. If more than one band disappears by peptide/antigen competition then those bands have the antigenic determinants and could be considered either fragments of the large antigen or multimer. 1. Determine the amount of antibody that is needed for 1 strip (e.g., 2 ml). For example, an antibody has given desired bands at 1:1000 dilution. So you will need 1 ul/ml antibody (2-ul antibody for 2-ml antibody solution). If an antibody were used at 1:5000 dilution then you would only need 0.2 ul/ml (use 2 ul of 1:10 dilution for better accuracy). 2. Take 2-ul antibody (or as needed) in 100 ul saline/PBS. Make 2 tubes. Add antigen/peptide solution (10-50 ug peptide or antigen added in 10-100 ul). Add same volume of saline/PBS (no peptide/antigen) to the other tube labeled as -peptide or No peptide. Mix gently. 3. Incubate both tubes at 37oC for 1-2 hrs or 2-24 hrs at 4oC. 4. Centrifuge the tubes for 15 min at 4oC in a microfuge (10-15000 rpm) to pellet any immune complexes. Carefully remove the supernatant. If no visible pellet is seen than just leave approx. 5-10 ul at the bottom to avoid disturbing invisible immune complexes. If you do not centrifuge the solution, it may give high background. 5. After centrifugation, make up the volume of supt. to 2 ml (or what is necessary depending upon the initial antibody taken) with buffer (PBS-Tween with or without BSA or milk or any buffer that was used initially). Use both antibodies (with and without antigen/peptide) for Western blotting or immunolocalization. 4. Observe the bands/staining that disappears. We have tested ab4086 specificity by mixing it at 1/500 with 1µg/ml of peptide Histone H1.2 ab16936.

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    Abcam Scientific Support

    回复于 Apr 26 2005

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