Overview

  • Product name
    Anti-Histone H1.4 (phospho T18) antibody
    See all Histone H1.4 primary antibodies
  • Description
    Rabbit polyclonal to Histone H1.4 (phospho T18)
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, ICC/IF, ICCmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mouse, Rat, Cow, Pig
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H1, phosphorylated at T18.

    Read Abcam's proprietary immunogen policy (Peptide available as ab10138.)

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Storage buffer
    Preservative: 0.02% Sodium Azide
    Constituents: 1% BSA, PBS, pH 7.4
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

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.

Application Abreviews Notes
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.

Target

  • Function
    Histones H1 are necessary for the condensation of nucleosome chains into higher order structures.
  • Sequence similarities
    Belongs to the histone H1/H5 family.
    Contains 1 H15 (linker histone H1/H5 globular) domain.
  • Post-translational
    modifications
    Acetylated at Lys-26. Deacetylated at Lys-26 by SIRT1.
  • Cellular localization
    Nucleus. Chromosome.
  • Information by UniProt
  • Database links
  • Alternative names
    • 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

Images

  • 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: 

  • 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).

References

This product has been referenced in:
  • 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). Read more (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). Read more (PubMed: 24873882) »
See all 2 Publications for this product

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

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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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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