Overview

  • Product name
    Anti-Histone H1 (phospho T146) antibody
    See all Histone H1 primary antibodies
  • Description
    Rabbit polyclonal to Histone H1 (phospho T146)
  • Host species
    Rabbit
  • Specificity
    The accession number of the protein this antibody was raised against is NP_005312. This antibody is expected to recognise phospho T146 in H1.2, H1.3 (both 88% sequence identity with immunogen) and Human H1.4 (100% sequence identity with immunogen).
  • Tested applications
    Suitable for: WB, ICC/IF, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mouse
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 100 - 200 of Human Histone H1.4, phosphorylated at T146.

    Read Abcam's proprietary immunogen policy (Peptide available as ab10139.)

  • Positive control
    • HeLa Histone Preparation Nuclear Lysate - Colcemid-treated

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 ab3596 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 concentration.
IHC-P Use a concentration of 2 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

Images

  • Rabbit polyclonal to Histone H1 phospho T146 (1/1000) against recombinant Histone H1, incubated with (control) untreated insect cell 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).

    Using site-directed mutagenesis mutant Histone H1 proteins were made. Five phosphorylation cyclin/cdk phosphorylation concensus sites were mutated : T18, T146, T154, S172 and S187.

    Lanes 3-12 contain mutant histone H1 with only one wild-type cyclin/cdk phosphorylation concensus site (indicated in brakets).

    Lanes 13-14 contain mutant Histone H1 with all 5 sites mutated to Ala.

    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 

  • Rabbit polyclonal to Histone H1.4 (phospho T146) (1/1000).

    Human neuroblastoma cell line SK-N-SH cultured on coverslips were fixed in 4% paraformaldehye and then stained with ab3596 (green). The DNA stained with DAPI is shown in red. (100x magnification).

  • All lanes : Anti-Histone H1 (phospho T146) antibody (ab3596) at 1 µg/ml

    Lane 1 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treated
    Lane 2 : HeLa Histone Preparation Nuclear Lysate
    Lane 3 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treated (ab30751) with Histone H1 (phospho T146) peptide (ab10139) at 1 µg/ml
    Lane 4 : HeLa Histone Preparation Nuclear Lysate with Histone H1 (phospho T146) peptide (ab10139) at 1 µg/ml
    Lane 5 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treated (ab30751) with Human Histone H1 peptide (ab30741) at 1 µg/ml
    Lane 6 : HeLa Histone Preparation Nuclear Lysate with Human Histone H1 peptide (ab30741) at 1 µg/ml

    Lysates/proteins at 2.5 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Observed band size: 32 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 110 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 1 minute
  • ICC/IF image of ab3596 stained MCF7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3596, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) HeLa, Hek293 and HepG2 cells at 1µg/ml, and in 4% PFA fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml.
  • ab3596 (2µg/ml) staining histone H1 Phospho T146 in human colon using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining of smooth muscle alongside nuclear and cytoplasmic staining of myenteric plexus.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

References

This product has been referenced in:
  • Izquierdo-Bouldstridge A  et al. Histone H1 depletion triggers an interferon response in cancer cells via activation of heterochromatic repeats. Nucleic Acids Res 45:11622-11642 (2017). Read more (PubMed: 28977426) »
  • Mayor R  et al. Genome distribution of replication-independent histone H1 variants shows H1.0 associated with nucleolar domains and H1X associated with RNA polymerase II-enriched regions. J Biol Chem 290:7474-91 (2015). WB ; Human . Read more (PubMed: 25645921) »
See all 10 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Answer

Thank you for your call this morning. I have set up the free replacement of one vial, and will arrange a second pending your results.

For our records, can you please give me a few details of the samples you are running in the gel for blotting, and that you are staining for the ICC data? In particular, we are interested in the species, cell type, and method of fixation and permeabilization for the ICC.

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Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Arabidopsis thaliana Cell (Landsberg culture cell)
Specification
Landsberg culture cell
Fixative
Paraformaldehyde
Blocking step
BSA as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 3%

Dr. Peter McKeown

Verified customer

Submitted Apr 18 2007

Question
Answer

Thank you for forwarding such detailed information on how this researcher has used the antibody. I have a number of suggestions which I hope will be helpful to them - I'm confident of the quality of the antibody, it's just a case of our better explaining how it has been tested and making recommendations to the researcher's protocol. May I remind you though that we require feedback to be submitted via our website or via an e-mail (rather than attached as a word document) - as explained by Dr Sarah Mardle, our tech support manager & Cheng Eng in our marketing team. The image on our datasheet explaining how this antibody has been tested in Western Blotting is not explained well and there are mistakes in the text. I will have somebody correct & improve the figure legend. In this experiment, recombinant histone H1 was incubated with cell lysates either with or without active cyclin E / CDK2 complexes. When the kinase (active cyclin E / CDK2) is present, you would expect threonine 146 to be phosphorylated, when absent, you would expect no phosphorylation. There is a band in lane 2 where wild type histone H1 was used and the kinase is present. T146 is phosphorylated and thus recognised by the antibody. As you'd expect, the band is absent in lane 1 when the kinase is absent. The rest of the lanes feature different mutant histone H1 recombinant proteins and the only time a band is observed is where there's a threonine at AA postion 146 and the kinase is present. This experiment demonstrates that the antibody does not react with phosphorylated or unphosphorylated threonine residues at AA positions 18, 154 & 172 & 187. In this experiment to demonstrate specificity, the antibody was used at a dilution of 1:1000 - it's important to note that in this experiment it's recognising recombinant Histone protein that will be present in much more abundant quantities than your researcher will find at endogenous levels in the cell lysates or nuclear extracts they are using. Furthermore, the levels of histone H1.4 phosphorylated at the threonine 146 will be even lower in their endogenous samples. This could very well explain why your customer sees no bands with Huh7 & Cos7 whole cell lysates but does see a very weak band with purified H1.4 protein from HeLa cells. I'd recommend trying the antibody at a much lower dilution with the purified H1.4 protein from the HeLa cells - 1:100 rather than 1:1000. There are as yet no publications that feature the use of this antibody, but I hope that the above explanation of the experiment featured on our datasheet gives the researcher a better understanding of how they can get this product to work in their hands. Do let us know if they need any more help.

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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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