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    products/primary-antibodies/huntingtin-antibody-ep867y-ab45169.pdf

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Neuroscience Neurology process Neurodegenerative disease Huntington's disease
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Huntingtin antibody [EP867Y] (ab45169)

  • Datasheet
  • SDS
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Western blot - Anti-Huntingtin antibody [EP867Y] (ab45169)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Huntingtin antibody [EP867Y] (ab45169)
  • Western blot - Anti-Huntingtin antibody [EP867Y] (ab45169)
  • Flow Cytometry (Intracellular) - Anti-Huntingtin antibody [EP867Y] (ab45169)
  • Immunocytochemistry/ Immunofluorescence - Anti-Huntingtin antibody [EP867Y] (ab45169)
  • Western blot - Anti-Huntingtin antibody [EP867Y] (ab45169)
  • Anti-Huntingtin antibody [EP867Y] (ab45169)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EP867Y] to Huntingtin
  • Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Human

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 647 APC Carrier Free PE

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Recombinant Human Huntingtin protein (Tagged) (ab112300)
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Overview

  • Product name

    Anti-Huntingtin antibody [EP867Y]
    See all Huntingtin primary antibodies
  • Description

    Rabbit monoclonal [EP867Y] to Huntingtin
  • Host species

    Rabbit
  • Tested applications

    Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IFmore details
    Unsuitable for: IP
  • Species reactivity

    Reacts with: Mouse, Human
    Predicted to work with: Rat
  • Immunogen

    Synthetic peptide within Human Huntingtin aa 550-650. The exact sequence is proprietary. Corresponding to residues specific to the apopain cleavage site.

  • Positive control

    • ICC/IF: SK-N-SH cells. WB: HAP1, SH-SY5Y and HeLa whole cell lysates. IHC-P: Human brain tissue. Flow Cyt (intra): SH-SY5Y cells.
  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 9% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA, 50% Tissue culture supernatant
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EP867Y
  • Isotype

    IgG
  • Research areas

    • Neuroscience
    • Neurology process
    • Neurodegenerative disease
    • Huntington's disease

Associated products

  • Alternative Versions

    • Alexa Fluor® 488 Anti-Huntingtin antibody [EP867Y] (ab206048)
    • Alexa Fluor® 647 Anti-Huntingtin antibody [EP867Y] (ab206049)
    • APC Anti-Huntingtin antibody [EP867Y] (ab224969)
    • PE Anti-Huntingtin antibody [EP867Y] (ab224970)
    • Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (ab96899)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

    • Human HTT (Huntingtin) knockout HeLa cell line (ab265976)
  • KO cell lysates

    • Human HTT (Huntingtin) knockout HeLa cell lysate (ab256946)
  • Recombinant Protein

    • Recombinant Human Huntingtin protein (Tagged) (ab112300)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab45169 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
Flow Cyt (Intra)
1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

WB
1/10000. Predicted molecular weight: 348 kDa.
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF
1/50 - 1/100.
Notes
Flow Cyt (Intra)
1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

WB
1/10000. Predicted molecular weight: 348 kDa.
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF
1/50 - 1/100.
Application notes
Is unsuitable for IP.

Target

  • Function

    May play a role in microtubule-mediated transport or vesicle function.
  • Tissue specificity

    Expressed in the brain cortex (at protein level). Widely expressed with the highest level of expression in the brain (nerve fibers, varicosities, and nerve endings). In the brain, the regions where it can be mainly found are the cerebellar cortex, the neocortex, the striatum, and the hippocampal formation.
  • Involvement in disease

    Defects in HTT are the cause of Huntington disease (HD) [MIM:143100]. HD is an autosomal dominant neurodegenerative disorder characterized by involuntary movements (chorea), general motor impairment, psychiatric disorders and dementia. Onset of the disease occurs usually in the third or fourth decade of life and symptoms progressively worsen leading to death in 10 to 20 years. Onset and clinical course depend on the degree of poly-Gln repeat expansion, longer expansions resulting in earlier onset and more severe clinical manifestations. HD affects 1 in 10,000 individuals of European origin. Neuropathology of Huntington disease displays a distinctive pattern with loss of neurons, especially in the caudate and putamen (striatum).
  • Sequence similarities

    Belongs to the huntingtin family.
    Contains 10 HEAT repeats.
  • Domain

    The N-terminal Gln-rich and Pro-rich domain has great conformational flexibility and is likely to exist in a fluctuating equilibrium of alpha-helical, random coil, and extended conformations.
  • Post-translational
    modifications

    Cleaved by apopain downstream of the polyglutamine stretch. The resulting N-terminal fragment is cytotoxic and provokes apoptosis.
    Forms with expanded polyglutamine expansion are specifically ubiquitinated by SYVN1, which promotes their proteasomal degradation.
  • Cellular localization

    Cytoplasm. Nucleus. The mutant Huntingtin protein colocalizes with AKAP8L in the nuclear matrix of Huntington's disease neurons.
  • Target information above from: UniProt accession P42858 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 3064 Human
    • Entrez Gene: 15194 Mouse
    • Entrez Gene: 29424 Rat
    • Omim: 143100 Human
    • Omim: 613004 Human
    • SwissProt: P42858 Human
    • SwissProt: P42859 Mouse
    • SwissProt: P51111 Rat
    • Unigene: 518450 Human
    • Unigene: 209071 Mouse
    • Unigene: 482929 Mouse
    • Unigene: 11193 Rat
    see all
  • Alternative names

    • AI256365 antibody
    • C430023I11Rik antibody
    • HD antibody
    • HD protein antibody
    • HD_HUMAN antibody
    • HDH antibody
    • HTT antibody
    • Huntingtin antibody
    • HUNTINGTON CHOREA antibody
    • Huntington disease protein antibody
    • Huntington's disease protein homolog antibody
    • IT 15 antibody
    • IT15 antibody
    • OTTMUSP00000026909 antibody
    • ZHD antibody
    see all

Images

  • Western blot - Anti-Huntingtin antibody [EP867Y] (ab45169)
    Western blot - Anti-Huntingtin antibody [EP867Y] (ab45169)
    All lanes : Anti-Huntingtin antibody [EP867Y] (ab45169) at 1/10000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : HTT knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 348 kDa
    Observed band size: 348 kDa



    Lanes 1- 2: Merged signal (red and green). Green - ab45169 observed at 348 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

     ab45169 was shown to react with Huntingtin in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265976 (knockout cell lysate ab256946) was used. Wild-type HeLa and HTT knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab45169 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Huntingtin antibody [EP867Y] (ab45169)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Huntingtin antibody [EP867Y] (ab45169)

    Ab45169 staining human Huntingtin in human brain tissue by immunohistochemistry using paraffin embedded tissue.

    Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

  • Western blot - Anti-Huntingtin antibody [EP867Y] (ab45169)
    Western blot - Anti-Huntingtin antibody [EP867Y] (ab45169)
    All lanes : Anti-Huntingtin antibody [EP867Y] (ab45169) at 1/10000 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : Huntingtin knockout HAP1 whole cell lysate
    Lane 3 : SH-SY5Y whole cell lysate
    Lane 4 : HeLa whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 348 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab45169 observed at 348 kDa. Red - loading control, ab18058, observed at 130 kDa.

    ab45169 was shown to specifically recognize Huntingtin in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when Huntingtin knockout samples were examined. Wild-type and Huntingtin knockout samples were subjected to SDS-PAGE. ab45169 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/10,000 dilution and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

  • Flow Cytometry (Intracellular) - Anti-Huntingtin antibody [EP867Y] (ab45169)
    Flow Cytometry (Intracellular) - Anti-Huntingtin antibody [EP867Y] (ab45169)

    Overlay histogram showing SH-SY5Y cells stained with ab45169 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab45169, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10^6 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Tween used under the same conditions. 

     

     

  • Immunocytochemistry/ Immunofluorescence - Anti-Huntingtin antibody [EP867Y] (ab45169)
    Immunocytochemistry/ Immunofluorescence - Anti-Huntingtin antibody [EP867Y] (ab45169)

    ICC/IF image of ab45169 stained SKNSH 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 (ab45169, 1/200 dilution) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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.

  • Western blot - Anti-Huntingtin antibody [EP867Y] (ab45169)
    Western blot - Anti-Huntingtin antibody [EP867Y] (ab45169)
    Anti-Huntingtin antibody [EP867Y] (ab45169) at 1/10000 dilution + SH-SY-5Y cell lysate

    Predicted band size: 348 kDa
    Observed band size: 300 kDa why is the actual band size different from the predicted?

  • Anti-Huntingtin antibody [EP867Y] (ab45169)
    Anti-Huntingtin antibody [EP867Y] (ab45169)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (5)

Publishing research using ab45169? Please let us know so that we can cite the reference in this datasheet.

ab45169 has been referenced in 5 publications.

  • Hartlage-Rübsamen M  et al. Endogenous mouse huntingtin is highly abundant in cranial nerve nuclei, co-aggregates to Abeta plaques and is induced in reactive astrocytes in a transgenic mouse model of Alzheimer's disease. Acta Neuropathol Commun 7:79 (2019). PubMed: 31109380
  • Lin CL  et al. Oral treatment with herbal formula B307 alleviates cardiac failure in aging R6/2 mice with Huntington's disease via suppressing oxidative stress, inflammation, and apoptosis. Clin Interv Aging 10:1173-87 (2015). IHC-P ; Mouse . PubMed: 26229452
  • Czeredys M  et al. Expression of genes encoding the calcium signalosome in cellular and transgenic models of Huntington's disease. Front Mol Neurosci 6:42 (2013). WB ; Human . PubMed: 24324398
  • Clemen CS  et al. Strumpellin is a novel valosin-containing protein binding partner linking hereditary spastic paraplegia to protein aggregation diseases. Brain 133:2920-41 (2010). PubMed: 20833645
  • Cho KJ  et al. Inhibition of apoptosis signal-regulating kinase 1 reduces endoplasmic reticulum stress and nuclear huntingtin fragments in a mouse model of Huntington disease. Neuroscience 163:1128-34 (2009). PubMed: 19646509

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