Anti-Histone H1 (phospho T146) antibody (ab3596)

Overview

  • Product nameAnti-Histone H1 (phospho T146) antibodySee all Histone H1 primary antibodies ...
  • Description
    Rabbit polyclonal to Histone H1 (phospho T146)
  • SpecificityThe 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 applicationsWB, 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.

    (Peptide available as ab10139.)

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

Properties

  • FormLiquid
  • Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Storage bufferPreservative: 0.02% Sodium Azide
    Constituents: 1% BSA, PBS, pH 7.4
  • Concentration information loading...
  • PurityImmunogen affinity purified
  • Clonality Polyclonal
  • IsotypeIgG
  • 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.Can be blocked with Histone H1 (phospho T146) peptide (ab10139).
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

Anti-Histone H1 (phospho T146) antibody 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  :  T18A, T146A, T154A, S187A (wt site at S172)
    Lanes 11-12:  T18A, T146A, T154A, S172A (wt site at S187)
    Lanes 13-14:  T18A, T146A, T154A, S172A, S187A.


    Lane two shows that Histone H1 is phosphorylated in the presence of cyclin E/ CDK2 complexes whilst in lane 1 the site is unphosphorylated and the ab doesn't recognise it.

    Lane 6 contains a mutated H1 histone where all of the phosphorylatable residues are mutated with the exception of T146. This shows that the antibody recognises T146 residue. The two bands appear to be running at different levels but we believe that this is due to curvature of the gel.

    Alejandro Contreras, Baylor College of Medicine

  • 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
    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 for Anti-Histone H1 (phospho T146) antibody (ab3596)

This product has been referenced in:
  • Absalon S  et al. MiR-26b, upregulated in Alzheimer's disease, activates cell cycle entry, tau-phosphorylation, and apoptosis in postmitotic neurons. J Neurosci 33:14645-59 (2013). Read more (PubMed: 24027266) »
  • Raghuram N  et al. Pin1 promotes histone H1 dephosphorylation and stabilizes its binding to chromatin. J Cell Biol 203:57-71 (2013). Read more (PubMed: 24100296) »

See all 8 Publications for this product

Product Wall

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 blottin...

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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%
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Dr. Peter McKeown

Verified customer

Submitted Apr 18 2007

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 explaini...

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