Anti-Histone H1 (phospho T146) antibody (ab3596)
Key features and details
- Rabbit polyclonal to Histone H1 (phospho T146)
- Suitable for: WB, ICC/IF, IHC-P
- Reacts with: Human
- Isotype: IgG
Overview
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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
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Immunogen
Synthetic peptide corresponding to Human Histone H1 aa 100-200 (C terminal) conjugated to keyhole limpet haemocyanin.
(Peptide available asab10139) -
Positive control
- HeLa Histone Preparation Nuclear Lysate - Colcemid-treated
Properties
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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
pH: 7.40
Preservative: 0.02% Sodium azide
Constituent: PBS
Batches of this product that have a concentration
Concentration information loading...Purity
Immunogen affinity purifiedClonality
PolyclonalIsotype
IgGResearch areas
Associated products
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Compatible Secondaries
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Isotype control
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Recombinant Protein
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
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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
modificationsAcetylated at Lys-26. Deacetylated at Lys-26 by SIRT1. -
Cellular localization
Nucleus. Chromosome. - Information by UniProt
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Database links
- Entrez Gene: 3008 Human
- Entrez Gene: 50709 Mouse
- Omim: 142711 Human
- SwissProt: P10412 Human
- SwissProt: P16401 Human
- SwissProt: P43274 Mouse
- SwissProt: P43276 Mouse
- Unigene: 131956 Human
see all -
Alternative names
- H1 histone family antibody
- H14_HUMAN antibody
- H1F5 antibody
see all
Images
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Western blot - Anti-Histone H1 (phospho T146) antibody (ab3596)This image is courtesy of Alejandro Contreras, Baylor College of Medicine
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 -
Immunocytochemistry/ Immunofluorescence - Anti-Histone H1 (phospho T146) antibody (ab3596)This image is courtesy of Dr Michael Hendzel's laboratoryRabbit 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).
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Western blot - Anti-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 (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 -
Immunocytochemistry/ Immunofluorescence - Anti-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) - Anti-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.
Protocols
Datasheets and documents
References (12)
ab3596 has been referenced in 12 publications.
- Li YH et al. MTA1 Promotes Hepatocellular Carcinoma Progression by Downregulation of DNA-PK-Mediated H1.2T146 Phosphorylation. Front Oncol 10:567 (2020). PubMed: 32435614
- 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). 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 . PubMed: 25645921
- 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
- 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). PubMed: 24027266
Images
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Western blot - Anti-Histone H1 (phospho T146) antibody (ab3596) This image is courtesy of Alejandro Contreras, Baylor College of Medicine
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 -
Immunocytochemistry/ Immunofluorescence - Anti-Histone H1 (phospho T146) antibody (ab3596) This image is courtesy of 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 - Anti-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 (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
-
Immunocytochemistry/ Immunofluorescence - Anti-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) - Anti-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.
