Anti-Histone H1.4 (phospho T18) 抗体 (ab3595)

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Key features and details

  • Rabbit polyclonal to Histone H1.4 (phospho T18)
  • Suitable for: WB, ICC/IF, ICC
  • Reacts with: Human
  • Isotype: IgG

製品の概要

  • 製品名

    Anti-Histone H1.4 (phospho T18) antibody
    Histone H1.4 一次抗体 製品一覧

  • 製品の詳細

    Rabbit polyclonal to Histone H1.4 (phospho T18)

  • 由来種

    Rabbit

  • アプリケーション

    適用あり: WB, ICC/IF, ICCmore details

  • 種交差性

    交差種: Human
    交差が予測される動物種: Mouse, Rat, Cow, Pig

  • 免疫原

    Synthetic peptide corresponding to Human Histone H1.4 aa 1-100 conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab10138)

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

    pH: 7.20
    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...

  • 精製度

    Immunogen affinity purified

  • ポリ/モノ

    ポリクローナル

  • アイソタイプ

    IgG

  • 研究分野

アプリケーション

Our Abpromise guarantee covers the use of ab3595 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
WB 1/1000. Detects a band of approximately 32 kDa.
ICC/IF Use at an assay dependent dilution.
ICC Use at an assay dependent dilution.

ターゲット情報

  • 機能

    Histones H1 are necessary for the condensation of nucleosome chains into higher order structures.

  • 配列類似性

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

  • 翻訳後修飾

    Acetylated at Lys-26. Deacetylated at Lys-26 by SIRT1.

  • 細胞内局在

    Nucleus. Chromosome.
  • Information by UniProt

  • 参照データベース

    • 別名

      • H1 histone family member 4 antibody
      • H1.4 antibody
      • H14_HUMAN antibody
      • H1E antibody
      • H1F4 antibody
      • Hist1h1e antibody
      • Histone 1 H1e antibody
      • Histone cluster 1 H1e antibody
      • Histone H1 antibody
      • Histone H1.4 antibody
      • Histone H1B antibody
      • MGC116819 antibody
      see all

    画像

    • Western blot - Anti-Histone H1.4 (phospho T18) antibody (ab3595)
      Western blot - Anti-Histone H1.4 (phospho T18) antibody (ab3595)This image is courtesy of Alejandro Contreras, Baylor College of Medicine

      Rabbit polyclonal to Histone H1 phospho T18 (1/1000) against recombinant Histone H1, incubated with control lysate (lanes 1, 3, 5, 7, 9, 11, 13) or insect cell lysate containing active cyclin E / CDK2 complexes (lanes 2, 4, 6, 8, 10, 12, 14).

      Mutant Histone H1 proteins were made by site-directed mutagenesis of 5 phosphorylation cyclin/cdk phosphorylation concensus sites (T18, T146, T154, S172, S187). Lanes 3-12 contain mutant histone H1 with only one wild-type cyclin/cdk phosphorylation concensus site and lanes 13-14 containst mutant Histone H1 with all 5 sites mutated to Alanine.

      Lanes 1-2    :  wt Histone H1
      Lanes 3-4    :  T146A, T154A, S172A, S187A (wt site at T18)
      Lanes 5-6    :  T18A, T154A, S172A, S187A (wt site at T146)
      Lanes 7-8    :  T18A, T146A, S172A, S187A (wt site at T154)
      Lanes 9-10  :  T18A, T146A, T154A, S187A (wt site at S172)
      Lanes 11-12: 

    • Immunocytochemistry/ Immunofluorescence - Anti-Histone H1.4 (phospho T18) antibody (ab3595)
      Immunocytochemistry/ Immunofluorescence - Anti-Histone H1.4 (phospho T18) antibody (ab3595)This image is courtesy of Dr Michael Hendzels laboratory

      Rabbit polyclonal to Histone H1 (phospho T18) (1/25).

      Human neuroblastoma cell line SK-N-SH cultured on coverslips were fixed in 4% paraformaldehye and then stained with ab3595 (green). The DNA stained with DAPI is shown in red. (100x magnification).

    参考文献 (2)

    ab3595 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。

    ab3595 は 2 報の論文で使用されています。

    • Chen Y  et al. Quantitative Mass Spectrometry Reveals that Intact Histone H1 Phosphorylations are Variant Specific and Exhibit Single Molecule Hierarchical Dependence. Mol Cell Proteomics N/A:N/A (2015). PubMed: 26209608
    • Terme JM  et al. Dynamics and dispensability of variant-specific histone H1 Lys-26/Ser-27 and Thr-165 post-translational modifications. FEBS Lett 588:2353-62 (2014). PubMed: 24873882

    レビューと Q&A

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    1-2 of 2 Abreviews or Q&A

    Question

    Myself and an RA here have had no success using the anti-phosphoH1(T18) antibody against and acid-soluble HeLa fraction that contains all histones and should contain this phosphorylated form of H1. We noticed that there have been similar complaints about this particular lot of antibody. If possible, please send us a replacement vial of the same antibody from a diferent lot. The antibody was purchased on about December 7, 2004. Below is a summary of the order. Order Summary Information Order Number . 379617 OP Vendor Number. 281933 ABCAM LIMITED (USD) Address Number 963 INVITROGEN VAN ALLEN

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    Answer

    Thank you for your email and I'm sorry to hear that you are experiencing difficulty with ab3595. It would be very helpful if you could provide me with some additional details regarding your protocol. This will help us to investigate this matter as quickly as possible. Thank you again, and I look forward to hearing from you. 1. Please describe the problem (high background, wrong band size, more bands, no band etc). 2. On what material are you testing the antibody in WB? •Species? •Cell extract/ Nuclear extract? •Purified protein? •Recombinant protein? 3. How much protein did you load? •How did you prepare the lysate for the analysis (protease inhibitors etc)? •Did you heat the samples? 4. Primary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? 5. Secondary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? •Do you know whether the problems you are experiencing come from the secondary? 6. What detection method are you using? 7. Background bands •Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control) •Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4°C with agitation) •Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) •At what size are the bands migrating? Could they be degradation products of your target? •Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 8. Optimization attempts •How many times have you tried the Western? •Do you obtain the same results every time e.g. are background bands always in the same place? •What steps have you altered? 9. Did you apply positive and negative controls along with the samples? Please specify.

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    Question

    ANTIBODY CODE ab3595 BATCH NUMBER 33274 DESCRIPTION OF THE PROBLEM No signal or weak signal. In fact, there is almost no background which makes me suspect that the tube we received contains little or no IgG SAMPLE acid extracted whole histones and RP-HPLC purified H1 from asynchronous and colchicine arrested HeLa) PRIMARY ANTIBODY 1/1000 in TBST (TBS with 0.1 % Tween 20), 2 hours at RT or 4C overnight SECONDARY ANTIBODY Amersham donkey anti-rabbit IgG HRP conjugate, 1/5000 in TBST, 90 minutes followed by 5 washes in TBST DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED We identified phospho-T18 in the same colchicine arrested sample by tandem MS. This sample was positive in parallel blots probed with your antisera to phospho-T146 H1 (ab3596, lot 33252). ANTIBODY STORAGE CONDITIONS aliquots stored at -80C SAMPLE PREPARATION A complete spectrum of protease and phosphatase inhibitors was used duirng chromatin and histone isolation. Samples were heated for 3 minutes at 95C in SDS sample buffer just before loading on gel. AMOUNT OF PROTEIN LOADED 1-2 ug with respect to H1 ELECTROPHORESIS/GEL CONDITIONS reducing SDS PAGE (12% gel) TRANSFER AND BLOCKING CONDITIONS semi-dry transfer (90 minutes) to PVDF or nitrocellulose, block with 5% milk powder in TBS for 2 hours at RT HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 8 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? We have compared nitro vs PVDF and tried using the primary to 1/200. We also tried blocking with BSA. The absence of background in lengthy ECL exposures is significant. ADDITIONAL NOTES Since we know our sample contains phospho-T18 molecules by mass spec and since your phospho T146 antibody works well used in parallel, either the antibody isn't any good or we received a bad lot. It seems unlikely that ab3595 was denatured in transit since we received both ab3595 and ab3596 in the same shipment.

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    Answer

    Thank you for your e-mail. We are very sorry to hear that you are having problem with this antibody. We do not have problem with this antibody and this is the first complaint we have received so far. Certainly, we can send you a new vial as a replacement - free of charge. Please do let us know how you are getting on with this vial.

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