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-P more 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 controlHeLa 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
  • Epigenetics and Nuclear Signaling
  • Histones
  • H1
  • Phosphorylated

Applications

Target

• FunctionHistones H1 are necessary for the condensation of nucleosome chains into higher order structures.
• Sequence similaritiesBelongs to the histone H1/H5 family.
  Contains 1 H15 (linker histone H1/H5 globular) domain.
• Post-translational
  modificationsAcetylated at Lys-26. Deacetylated at Lys-26 by SIRT1.
• Cellular localizationNucleus. Chromosome.

Target information above from: UniProt accession P10412 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
• Database links
  • Entrez Gene: 3008 Human
  • Entrez Gene: 50709 Mouse
  • Omim: 142711 Human
  • SwissProt: P16401 Human
  • SwissProt: P10412 Human
  • SwissProt: P43276 Mouse
  • SwissProt: P43274 Mouse
  • Unigene: 131956 Human

  • Unigene: 248133 Human
  • Unigene: 221314 Mouse
  • Unigene: 170587 Mouse

  see all

• Alternative names
  H1 histone family antibodyH14_HUMAN antibodyH1F4 antibody

  H1F5 antibodyHist1h1e antibodyHistone H1.4 antibodyHistone H1a antibodyHistone H1b antibody

  see all

Anti-Histone H1 (phospho T146) antibody images

• 

  Western blot - Histone H1 (phospho T146) antibody (ab3596)

  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

• 

  Immunofluorescence - Histone H1.4 (phospho T146) antibody (ab3596)Dr Michael Hendzel's laboratory

  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).

• 

  Western blot - Histone H1 (phospho T146) antibody (ab3596)
  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 peptide - phospho T146 (ab10139) at 1 µg/ml
  Lane 4 : HeLa Histone Preparation Nuclear Lysate with Histone H1 peptide - phospho T146 (ab10139) at 1 µg/ml
  Lane 5 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treated (ab30751) with Histone H1 peptide (ab30741) at 1 µg/ml
  Lane 6 : HeLa Histone Preparation Nuclear Lysate with 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
• 

  Immunocytochemistry/ Immunofluorescence - Histone H1 (phospho T146) antibody (ab3596)
  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.
• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Histone H1 (phospho T146) antibody (ab3596)

  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.